Enzymes and applications thereof

ABSTRACT

There is provided SHC/HAC derivatives, amino acid sequences comprising the SHC/HAC derivatives, nucleotide sequences encoding the SHC/HAC derivatives, vectors comprising nucleotide sequences encoding the SHC/HAC derivatives, recombinant host cells comprising nucleotide sequences encoding the SHC/HAC derivatives and applications of the recombinant host cells comprising either SHC/HAC derivatives or WT SHC/HAC enzymes in methods to prepare (−)-Ambrox and SHC/HAC enzymes in methods to prepare (−)-Ambrox.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 15/568,945 filed onOct. 24, 2017, now U.S. Pat. No. 10,472,655, which is a national stageapplication of International Application No. PCT/EP2016/058987, filedApr. 22, 2016, which claims priority from Great Britain PatentApplication No. 1507207.7, filed Apr. 24, 2015. Applicant incorporatesby reference the entirety of each of the foregoing documents herein, andclaims all available priority benefit to each of the above applications.

FIELD OF THE INVENTION

The present invention relates to Squalene Hopene Cyclase/HomofarnesolAmbrox Cyclase (SHC/HAC) derivative enzymes which have been modifiedwith respect to a reference SHC/HAC protein, amino acid sequencescomprising the SHC/HAC derivative enzymes, nucleotide sequences encodingthe SHC/HAC derivatives, vectors comprising nucleotide sequencesencoding the SHC/HAC derivatives and recombinant host cells comprisingnucleotide sequences encoding the SHC/HAC derivatives. The presentinvention also relates to means for functionally expressing nucleotidesequences encoding SHC/HAC derivatives and methods of using recombinantmicroorganisms comprising nucleotide sequences encoding SHC/HACderivatives and WT SHC/HAC to make Ambrox, preferably (−)-Ambrox.

BACKGROUND OF THE INVENTION

Squalene Hopene Cyclases (SHC, EC 5.4.99.17) are membrane-boundprokaryotic enzymes which act as biocatalysts for the cyclisation of thelinear triterpenoid squalene to hopene and hopanol. Earlier SHC workfocused on the characterisation of the SHC of the thermophilic andacidophilic bacterium Alicyclobacillus acidocaldarius (formerly Bacillusacidocaldarius) (see Neumann & Simon 1986, Biol Chem Hoppe-Seyler 367,723-729; Seckler & Poralla 1986, Biochem Biophys Act 356-363 and Ochs etal 1990, J Bacteriol 174, 298-302). However, more recently, other SHCsfrom Zymomonas mobilis and Bradyrhizobium japonicum have been purifiedand characterized in terms of their natural (eg. squalene) andnon-natural substrates (eg. homofarnesol and citral) (see for example,WO 2010/139710, WO 2012/066059 and Seitz et al 2012. J. MolecularCatalysis B: Enzymatic 84, 72-77).

Earlier work by Neumann and Simon (1986—as cited above) disclosed thathomofarnesol is an additional substrate for Alicyclobacillusacidocaldarius SHC (AacSHC). However, the cyclisation rate of thenon-natural homofarnesol by the purified AacSHC taught by Neumann andSimon (1986) was reported at only 3% of the cyclisation rate for thenatural substrate squalene. The rate of formation of Ambrox (product 2b)increased with the concentration of homofarnesol (product 1b) from 0.25mM to 2.0 mM and declined slightly in the presence of 4 mM of product1b. The difference m cyclisation rates may be attributed in part to thefact that the natural SHC substrate squalene is twice the size (a C30carbon compound) of the non-natural homofarnesol which is a C16 carboncompound.

(JP2009060799—Kao) also discloses a method for producing Ambrox fromhomofarnesol using an SHC from A. acidocadarius. Whilst JP2009060799teaches the possibility of using microorganisms comprising SHC for thesynthesis of Ambrox, JP2009060799 only discloses the production ofAmbrox from homofarnesol using an SHC liquid extract prepared from arecombinant microorganism expressing the SHC gene and not by means ofwhole recombinant microbial cells expressing the SHC gene. The percentconversion of homofarnesol to Ambrox using an SHC liquid extract wasreported as 17.5% when carried out at a temperature of 60° C. for 14hour at pH 5.2-6.0 but only as 6.8% when carried out at a pH of 6.6. Thepercent conversion of 3E, 7E-homofarnesol to Ambrox using an SHC liquidextract at 60° C. at pH 5.6 for 64 hours was reported as 63% when a 0.2%homofarnesol (2 g/l) substrate concentration is used.

WO 2010/139719A2 and its US equivalent (US2012/0135477A1) describe atleast three SHC enzyme extracts with homofarnesol to Ambrox cyclaseactivity. The Zymomonas mobilis (Zmo) SHC and the Bradyrhizobiumjaponicum (Bjp) SHC enzymes are reported to show homofarnesol conversionrates of 41% at 16 h of reaction and 22% respectively when a 10 mM (2.36g/l) homofarnesol concentration was used while the conversion rate forAacSHC was reported to be only 1.2% (presumably at the same homofarnesolconcentration) but no experimental details are provided. The ZmoSHC andBjpSHC enzyme extracts were prepared from a recombinant microorganismexpressing the SHC gene by disrupting the E. coli host cells producingthe SHC enzymes and separating the soluble SHC fractions.

Seitz et al (2012—as cited above) reports on the functional expressionand biochemical characterisation of three SHC enzymes, two from Z.mobilis (ZmoSHC1 and ZmoSHC2) and one from A. acidocaldarius. It isreported that an “efficient” conversion (22.95%) of homofarnesol toAmbrox was observed using the wild-type ZmoSHC1 with no conversion ofhomofarnesol to Ambrox using WT ZmoSHC2 and a relatively low conversion(3.4%) of homofarnesol to Ambrox for AacSHC was found when a 10 mM (2.36g/l) homofarnesol concentration was used. The trend observed for therelatively low conversion of homofarnesol to Ambrox for AacSHC which wasin accord with the results of Neumann and Simon (1986—as cited above)and as disclosed in WO 2010/139719A2 as also discussed above. The threeSHC enzymes were used in a cell suspension format (through partialdisruption of host E. coli cells using freeze-thaw cycles) and aspartially purified membrane-bound fractions.

WO2012/066059 discloses mutants with cyclase-activity and the usethereof in a method for the biocatalytic cyclisation of terpenes, suchas, in particular, for producing isopulegol by the cyclisation ofcitronellal; to a method for producing menthol and methods for thebiocatalytic conversion of other compounds with terpene type structuralmotifs. Sequence alignment of various SHCs identified phenylalanine-486(F486) as a strongly conserved amino acid residue and a series ofsubstitution variants were generated in the Zymomonas mobilis SHCenzyme. Some of these substitutions led to the loss of activity, whileothers resulted in the formation of novel terpenoid product(isopulegols) from terpene substrates such as citronellal.

A report in a PhD thesis by Seitz in 2012 entitled “Characterization ofthe Substrate Specificity of Squalene-Hopene Cyclases (SHCs)” indicatesthat an F486Y mutation in ZmoSHC1 provided a diminished rate forhomofarnesol biotransformation of about 1.5 fold from 34.8% (WT ZmoSHC1)to 23.9% (mutant ZmoSHC1 F486Y). When the mutation equivalent (Y420C) inAacSHC was tested, it was presumed that the enzymatic activity towardsthe larger substrates would decrease and the activity towards smallersubstrates would rise. When the mutant was tested under the sameconditions as the wild-type and the enzymatic activities were compared,it was observed that the mutant did not show any conversion of thehomofarnesol substrate at all. Therefore it was concluded that Y420amino acid residue was crucial for the activity of AacSHC for allsubstrates. Other SHC site directed mutagenesis studies in the art (eg.Hoshino and Sato 2002, Chem Commun 291-301) were focused on the effectof mutations in highly conserved regions (eg. F601) and their effect onnatural substrates (i.e. squalene or squalene analogues) rather than onnon-natural substrates such as homofarnesol.

In summary, the limited disclosures in the art relating to bioconversionprocesses for the successful conversion of homofarnesol to Ambrox onlyrelate to relatively low concentrations/volumes of homofarnesolsubstrate (in the concentration range of from 0.25 mM to 2 mM to 10 mMor around 0.06 g/l to 2.36 g/l) using a wild-type SHC polypeptide withthe activity of a homofarnesol-Ambrox cyclase (HAC). The SHC enzymeswith HAC activity were either: (i) liquid extracts which were preparedeither by disrupting the E. coli host cells comprising the SHC enzymesand separating the insoluble and soluble SHC liquid fractions; (ii)partially purified membrane fractions; or (iii) recombinant whole cellsexpressing the WT SHC gene and producing the SHC enzyme for use inreactions for bioconverting homofarnesol to Ambrox using solubilizingagents including either: (i) Triton X-100 in the reaction mixture (seeNeumann and Simon 1986 as cited above, Seitz et al 2012 as cited above,JP2009060799); or (ii) taurodeoxycholate (as disclosed inUS2012/0135477A1).

Using these WT SHC extracts and/or whole recombinant microbial cellsexpressing the SHC gene, the homofarnesol to Ambrox conversion ratesobtained were found to vary depending on the source of the SHC enzyme,the amount of the homofarnesol starting material and the reactionconditions used. So far, a 100% percent conversion of homofarnesol toAmbrox using a wild-type SHC enzyme has not been achieved at thereported involved concentrations (0.06-2.36 g/l). In addition,preliminary investigations using SHC derivatives prepared using sitedirected mutagenesis studies have only provided negative (i.e. reducedhomofarnesol conversion rates) rather than positive (i.e. improvedconversion rates) results. In addition, only purified SHC enzymeextracts or SHC membrane bound fractions have been used in the publishedstudies or whole recombinant microbial cells expressing a WT SHC geneunder specific reaction conditions which use solubilizing agents such asTriton X-100 or taurodeoxycholate. There is no demonstration that arecombinant microorganism comprising either a WT or mutant SHC mightprovide a more efficient and cost effective bioconversion ofhomofarnesol to Ambrox using optimized reaction conditions. Accordingly,it is desired to improve the cited known processes for preparing Ambroxfrom homofarnesol by at least improving reaction velocity, specificity,yield, productivity and reducing costs (by, for example, simplifying theprocess using either recombinant whole microbial cells or by using a“one pot” process combining both biocatalyst production andbioconversion steps).

SUMMARY OF THE INVENTION

The present invention in various aspects provides SHC/HAC derivatives,amino acid sequences comprising the SHC/HAC derivative enzymes,nucleotide sequences encoding the SHC/HAC derivative enzymes, vectorscomprising nucleotide sequences encoding the SHC/HAC derivative enzymes,recombinant host cells comprising vectors with nucleotide sequencesencoding the SHC/HAC derivative enzymes and applications of therecombinant host cells comprising either SHC/HAC derivative enzymes orWT SHC/HAC enzymes when used under specific reaction conditions inmethods to prepare Ambrox materials comprising the Ambrox isomerdesignated (−)-Ambrox and Ambrox like molecules (as by products). Unlikethe disclosures in the art relating to AacSHC, the Applicant hasdemonstrated for the first time that a whole recombinant microorganismexpressing a SHC derivative gene can be used to bioconvert homofarnesolto Ambrox. In addition, a whole recombinant microorganism expressing aWT SHC gene and/or producing SHC enzymes can be used to bioconverthomofarnesol to Ambrox under specific reaction conditions not disclosedin the art.

It has also been surprisingly found that introducing up to five aminoacid alterations into the amino acid sequence of the WT SHC/HACreference sequence as disclosed herein leads to SHC/HAC derivativeenzymes with significantly improved homofarnesol to Ambrox conversionrates as compared with the unmodified SHC reference enzymes as disclosedherein. These novel SHC/HAC derivative enzymes are useful alone and incombination for the production of Ambrox materials, in particular(−)-Ambrox from homofarnesol substrates.

An additional surprising finding is that apart from one mutant (F601Y),the SHC derivative enzymes as disclosed herein typically comprisenon-conservative substitutions at amino acid residue positions in thenon-conserved part of the reference SHC polypeptide sequence. This is anunexpected finding as changes in the conserved region of an enzyme aremore likely to disrupt the function of an enzyme (at least in relationto its natural substrate) than changes in a non-conserved region of aprotein.

A further surprising finding is that the characterised SHC derivativeenzymes of the present disclosure perform optimally (on a non-naturalsubstrate such as homofarnesol) at about 35° C. rather than about 60° C.which is the usual reaction temperature for thermophilic microorganismssuch as AacSHC. The application of the SHC derivatives of the presentdisclosure in methods for preparing Ambrox from homofarnesol at lowerreaction temperatures has significant cost advantages for the Ambroxproduction cycle at an industrial scale.

Another advantage of the present invention is that the SHC derivativeenzymes of the present disclosure catalyse an efficient bioconversionprocess which when optimized with relatively high (eg. about 50 fold)homofarnesol substrate concentration compared to previously describedconcentrations in the prior art (eg. EEH at 125 g/l) can lead to 100%conversion of the homofarnesol substrate while the reference WT SHCprotein only converts about 10% of the same substrate even at a highconcentration of enzyme/cells. The disclosures in the cited prior artall relate to the use of purified membrane extracts comprising SHC orpurified SHC extracts (prepared from microorganisms expressing SHCgenes) or the use of recombinant microorganism expressing a WT SHC geneunder specific bioconversion reaction conditions (eg. using specificsolubilizing agents). Even then, a 100% homofarnesol conversion at muchlower EEH concentration is not reported. Also a “one pot” reaction wherein a first step the recombinant cells grow and produce the SHC enzymeand subsequently convert EEH to (−)-Ambrox in the same vessel has notbeen reported. A further advantage of the present invention is that therecombinant host cells producing the SHC derivative enzymes show highinitial reaction rates which allow the production of a high quantity ofproduct within a relatively short period of time while using onlyrelatively low amounts of biocatalyst. In short, the selection andefficient expression and application of recombinant microorganismscomprising either WT SHC/HAC or specific SHC/HAC derivative enzymesunder specific bioconversion reaction conditions leads to a moreefficient bioconversion process. The end product ((−)-Ambrox) can beseparated and easily purified. Unlike the cited art, the SHC/HACderivative enzymes are not used as a pure enzyme but are used in a wholecell context (as the biocatalyst) which is a more cost effective and amore user and environmentally friendly approach as no additional enzymepurification and isolation steps are required.

In summary, the present disclosure provides abioconversion/biotransformation method for making Ambrox in arecombinant microbial strain of which is: (i) economically attractive,(ii) environmentally friendly and (iii) leads to the selectiveproduction of (−)-Ambrox as a predominant compound which under selectivecrystallization conditions is effectively separated from otherby-products which do not contribute to the olfactive quality of the endproduct.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “SHC” means a Squalene Hopene Cyclase enzymefrom any of the sources listed in Tables 10-12. In preferredembodiments, the term SHC includes the Zymomonas mobilis SHC enzymes andthe Alicyclobacillus acidocaldarius SHC enzymes as disclosed in BASF WO2010/139719, US2012/01345477A1, Seitz et al (2012 as cited above) andSeitz (2012 PhD thesis as cited above). For ease of reference, thedesignation “AacSHC” is used for Alicyclobacillus acidocaldarius SHC andthe designation “ZmoSHC” is used for Zymomonas mobilis SHC and thedesignation “BjpSHC” is used for Bradyrhizobium japonicum SHC. Thepercent sequence identity for these sequences relating to WT AacSHC andeach other (which can vary depending on the algorithm used) is set outin Tables 18 and 19.

An alignment of WT SHC sequences prepared by Hoshino and Sato (2002 ascited above) indicates that multiple motifs were detected in all foursequences and consists of the core sequence Gln-X-X-X-Gly-X-Trp which isfound six times in the SHC sequences of both Z. mobilis and A.acidocaldarius (See FIG. 3 of Reipen et at 1995. Microbiology 141,155-161). Hoshino and Sato (2002 as cited above) report that aromaticamino acids are unusually abundant in SHCs and that two characteristicmotifs were noted in the SHCs, one is a QW motif represented by specificamino acid motifs [(K/R)(G/A)X2-3(F/Y/W)(L/IV)3X3QX2-5GXW] and thealternative is a DXDDTA motif. Wendt et a (1997, Science 277, 1811-1815and 1999, J Mol Biol 286, 175-187) reported on the X-ray structureanalysis of A. acidocaldarius SHC. The DXDDTA motif appears to correlatewith the SHC active site. Exemplary sequence alignments from the priorart are showing the recurring multiple motifs as provided in FIG. 2(from Hoshino and Sato (2002 as cited above)) and FIG. 3 (from Seitz PhDthesis (2012)) herein.

The reference (or wild-type) AacSHC protein as used herein refers to theAacSHC protein as disclosed in SEQ ID No. 1. The reference AacSHC enzymeof the present disclosure has the activity of a homofarnesol Ambroxcyclase (HAC) useful in the production of Ambrox derivatives through abiocatalytic reaction of SHC with a homofarnesol substrate. The mainreaction of the reference AacSHC is the cyclisation of a linear or anon-linear substrate such as homofarnesol to produce Ambrox.

Ambrox

As used herein, the term “Ambrox” includes (−)-Ambrox of formula (I) aswell as (−)-Ambrox in stereoisomerically pure form or in a mixture withat least one or more of the following molecules of formula (II), (IV)and/or (III).

(−)-Ambrox

(−)-Ambrox is known commercially as Ambrox (Firmenich), Ambroxan(Henkel) Ambrofix (Givaudan), Amberlvn (Quest), Cetalox Laevo(Finnenich), Ambennor (Aromor) and/or Norambrenolide Ether (Pacific).

(−)-Ambrox is an industrially important aroma compound and has been usedin the Fragrance industry for a long time. The special desirable sensorybenefits from (−)-Ambrox come from the (−) stereoisomer rather than the(+) one. The odour of the (−) stereoisomer is described as musk-like,woody, warm or ambery whereas the (+)-Ambrox enantiomer has a relativelyweak odour note. The odour and odour thresholds for Ambrox like productsare also different. While various (−)-Ambrox enriched materials areavailable commercially, it is desirable to produce highly enriched(−)-Ambrox materials, ideally pure (−)-Ambrox.

Production of (−)-Ambrox

(−)-Ambrox can be produced from sclareolide according to the productionprocess as described below. Sclareol is a product extracted from thenatural plant clary sage. However, because a natural starting materialis used in this process, there are potential problems in that itinvolves a multistage reaction, its operation is circuitous, thequantity and stability of supply of starting material may not always besatisfactory, and the reaction may not be environmentally friendlybecause an oxidizing agent such as chromic acid or a permanganate isused in the step of (+)-sclareol oxidative degradation.

(−)-Ambrox is also synthesized from homofarnesol using different routes.By way of example, homofarnesol can be obtained by brominating,cyanating, and hydrolysing nerolidol to give homofarnesylic acid,followed by reduction. Alternatively, homofarnesol may be obtained fromfarnesol, farnesylchloride, beta-farnesene or other substrates.Beta-farnesene can be converted directly to E,E-homofarnesol (EEH) orindirectly to EEH via E,E-homofarnesate which is then converted to EEH.An overview on the production of (−)-Ambrox from different substratescan be found in US2012/0135477A1, WO 2010/139719, US2013.0273619A1, WO2013/156398A1 and the Seitz PhD thesis (2012 as cited above) andSchaefer 2011 (Chemie Unserer Zeit 45, 374-388).

Whilst homofarnesol may present as a mixture of four isomers, the(3Z,7Z), (3E,7Z), (3Z,7E) and (3E,7E) isomers, it seems from theliterature that (−)-Ambrox is only obtained from (3E,7E) homofarnesol(see Neumann and Simon (1986) as cited above). As used herein, areference to (3E,7E) homofarnesol is a reference to E,E-homofarnesolwhich is also designated as EEH.

US2012/0135477A1 reports on the conversion of (3Z,7E) to (−)-Ambroxusing ZmoSHC (SEQ ID No. 2) (see Examples 2-4) but according to thedisclosure in Schaefer (2011) (as cited above), (7E, 3Z) is onlyconverted to 9b-epi-Ambrox (i.e. compound III) as outlined above and notto (−)-Ambrox. As used herein, a reference to (3Z,7E) homofarnesol is areference to E, Z-homofarnesol which is also designated as EZH.

In some embodiments, preferably the homofarnesol starting materialcomprises a mixture of (3E,7E) and (3Z,7E), termed herein an EE:EZstereoisomeric mixture (particularly with reference to the Examples andTable 20.

An EE:EZ stereoisomeric mixture of homofarnesol has the CAS number of35826-67-6.

As the Examples demonstrate (eg, see Examples 5, 7, 9, 10, 11, 18, 19and 20), in certain embodiment, the homofarnesol feedstock/startingmaterial is a mixture of isomers.

Accordingly, in some embodiments, the homofarnesol starting material mayalso comprise a mixture of the four isomers EE:EZ:ZZ:ZE whichcorresponds with (3E,7E) and (3Z,7E), (3Z,7Z and 3E, 7Z).

In some embodiments, preferably the homofarnesol starting material isselected from one of more of the following groups: [(3Z,7Z), (3E,7Z),(3Z,7E) and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and/or[(3E,7E) and (3E,7Z)].

Preferably the homofarnesol starting material is selected from one ormore of the following groups-[(3E,7E), (3Z,7E)] and/or [(3Z,7E), (3E,7E)and (3E,7Z)].

Accordingly, in certain embodiments, the ratio of EEH:EZH is about100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09;90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19;80:20; 79:21; 78:22; 77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29;70:30; 69:31; 68:32; 67:33; 66:34; 65:35; 64:36; 63:37; 62:38; 61:39;60:40; 59:41; 58:42; 57:43; 56:44; 55:45; 54:46; 53:47; 52.48; 51:49; orabout 50:50.

In some embodiments preferably the homofarnesol starting materialcomprises >90% E,E-homofarnesol (EEH).

In other embodiments, the homofarnesol starting material comprises anEE:EZ weight ratio of 86:14.

In certain embodiments, the homofarnesol starting material comprises anEE:EZ weight ratio of 80:20.

In certain embodiments, the homofarnesol starting material comprises anEE:EZ weight ratio of 70:30.

In further embodiments, the homofarnesol starting material comprises anEE:EZ weight ratio of 69:31.

In some embodiments, the homofarnesol starting material consists of orconsists essentially of a mixture of the four isomers EE:EZ:ZZ:ZE whichcorresponds with (3E,7E) and (3Z,7E), (3Z,7Z) and (3E,7Z).

In some embodiments, preferably the homofarnesol starting materialconsists of or consists essentially of a mixture of the isomers selectedfrom one of more of the following groups: [(3Z,7Z), (3E,7Z), (3Z,7E) and(3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and/or [(3E,7E) and(3E,7Z)].

Preferably the homofarnesol starting material consists of or consistsessentially of a mixture of the isomers selected from one or more of thefollowing groups: [(3E,7E), (3Z,7E)] and/or [(3Z,7E), (3E,7E) and(3E,7Z)].

Accordingly, in certain embodiments, ratio of EEH:EZH isomers consistsof or consists essentially of a ratio of EEH:EZH of about 100:00; 99:01;98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11;88:12; 87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19; 80:20; 79:21;78:22; 77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29; 70:30; 69:31;68:32; 67:33; 66:34; 65:35; 64:36; 63:37; 62:38; 61:39; 60:40; 59:41;58:42; 57:43; 56:44; 55:45; 54:46; 53:47; 52:48; 51:49; or about 50:50.

In some embodiments preferably the homofarnesol starting materialsconsists of or consists essentially of >90% E,E-homofarnesol (EEH).

In other embodiments, the homofarnesol starting material consists of orconsists essentially of an EE:EZ weight ratio of 86:14.

In certain embodiments, the homofarnesol starting material consists ofor consists essentially of an EE:EZ weight ratio of 80:20.

In certain embodiments, the homofarnesol starting material consists ofor consists essentially of an EE:EZ weight ratio of 70:30.

In further embodiments, the homofarnesol starting material consists ofor consists essentially of an EE:EZ weight ratio of 69:31.

In embodiments of the present disclosure. Ambrox is produced using anSHC/HAC derivative enzyme.

SHC/HAC Derivative

As used herein, the term “SHC/HAC Derivative” means that the amino acidsequence of the SHC/HAC Derivative is a modified or variant amino acidsequence which is altered compared to the amino acid sequence of thereference (or wild-type) SHC sequence according to at least SEQ ID No. 1or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4. Generally SHC/HACderivatives comprise altered forms of SHC having at least one alterationthat modifies (eg. increases) the activity of the enzyme for itssubstrate (eg. EEH).

The SHC/HAC Derivatives of the present disclosure are tested for theirhomofarnesol Ambrox cyclase activity. Consequently, these SHC/HACDerivatives which convert homofarnesol to Ambrox are referred to hereinas HAC Derivatives as well as SHC Derivatives. Whilst exemplary SHC/HACderivatives have been provided for enzymes derived from Alicyclobacillusacidocaldarius, Zymomonas mobilis, Bradyrhizobium japonicum microbialstrain sources, the present disclosure also covers equivalent SHC/HACderivatives from other microbial strain sources which include but arenot limited to SHC/HAC enzymes from Methylococcus capsulatus, Frankiaalni, Acetobacter pasteurianum and Tetrahymena pyriformis (see forexample, WO 2010/139719, US2012/01345477, WO 2012/066059, and Tables10-12).

As used herein, the term “amino acid alteration” means an insertion ofone or more amino acids between two amino acids, a deletion of one ormore amino acids or a substitution (which may be conservative ornon-conservative) of one or more amino acids with one or more differentamino acids relative to the amino acid sequence of a reference aminoacid sequence (such as, for example, the wild-type (WT) amino acidsequence of SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No.4). The amino acid alterations can be easily identified by a comparisonof the amino acid sequences of the SHC/HAC derivative amino acidsequence with the amino acid sequence of the reference amino acidsequence (such as, for example, the wild-type (WT) amino acid sequenceof SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4).Exemplary WT SHC amino acid sequence alignments are provided in FIGS.1-4 and Tables 18 and 19.

Conservative amino acid substitutions may be made, for instance, on thebasis of similarity in polarity, charge, size, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theamino acid residues involved. The 20 naturally occurring amino acids asoutlined above can be grouped into the following six standard amino acidgroups:

(1) hydrophobic: Met, Ala, Val, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

Accordingly, as used herein, the term “conservative substitutions” meansan exchange of an amino acid by another amino acid listed within thesame group of the six standard amino acid groups shown above. Forexample, the exchange of Asp by Glu retains one negative charge in theso modified polypeptide. In addition, glycine and proline may besubstituted for one another based on their ability to disruptalpha-helices. Some preferred conservative substitutions within theabove six groups are exchanges within the following sub-groups: (i) Ala,Val, Leu and Ile, (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg;and (v) Tyr and Phe. Given the known genetic code, and recombinant andsynthetic DNA techniques, the skilled scientist readily can constructDNAs encoding the conservative amino acid variants.

As used herein, “non-conservative substitutions” or “non-conservativeamino acid exchanges” are defined as exchanges of an amino acid byanother amino acid listed in a different group of the six standard aminoacid groups (1) to (6) as shown above. Typically the SHC/HAC Derivativesof the present disclosure are prepared using non-conservativesubstitutions which alter the biological function (eg. HAC activity) ofthe disclosed SHC/HAC derivatives.

For ease of reference, the one-letter amino acid symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission are indicated asfollows. The three letter codes are also provided for referencepurposes.

One Three Amino add Letter Code Letter Code name A Ala Alanine C CysCysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine GGly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu LeucineM Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R ArgArginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan YTyr Tyrosine

Amino acid alterations such as amino acid substitutions may beintroduced using known protocols of recombinant gene technologyincluding PCR, gene cloning, site-directed mutagenesis of cDNA,transfection of host cells, and in-vitro transcription which may be usedto introduce such changes to the WT SHC sequence resulting in an SHC/HACderivative enzyme. The derivatives can then be screened for SHC/HACfunctional activity.

SHC/HAC Derivative Enzymes

The present invention provides an SHC/HAC derivative and describes anenzyme with homofarnesol Ambrox cyclase (HAC) activity which comprisesan amino acid sequence that has from about 1 to about 50 mutationsindependently selected from substitutions, deletions, or insertionsrelative to the amino acid sequence of the reference (or wild-type) SHCsequence according to at least SEQ ID No. 1 or SEQ ID No. 2 or SEQ IDNo. 3 or SEQ ID No. 4.

In various embodiments, the mutation or combination of mutationsenhances the activity of the SHC/HAC derivative for convertinghomofarnesol to Ambrox compared to reference SHC enzymes that do notshow this deletion/addition. Protein modeling as described herein may beused to guide such substitutions, deletions, or insertions in the SHCreference sequence. For example, a structural model of the SHC aminoacid sequence may be created using the coordinates for the AacSHC (asshown, for example in FIGS. 19 and 20). As demonstrated herein, such ahomology model is useful for directing improvement of SHC enzymes forconverting homofarnesol to (−)-Ambrox.

Thus, in various embodiments, the SHC/HAC derivative may have from about1 to about 45 mutations, about 1 to about 40 mutations, about 1 to about35 mutations, about 1 to about 30 mutations, about 1 to about 25mutations, from about 1 to about 20 mutations, about 1 to about 15mutations, about 1 to about 10 mutations, or from about 1 to about 5mutations relative to the amino acid sequence of the reference (orwild-type) SHC sequence according to at least SEQ ID No. 1 or SEQ ID No.2 or SEQ ID No. 3 or SEQ ID No. 4.

In various embodiments, the SHC/HAC derivative comprises a sequencehaving at least 5 or at least 10 mutations relative to the amino acidsequence of the reference (or wild-type) SHC sequence according to atleast SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 butnot more than about 20 or 30 mutations. In various embodiments, the SHCderivative may have about 1 mutation, about 2 mutations, about 3mutations, about 4 mutations, about 5 mutations, about 6 mutations,about 7 mutations, about 8 mutations, about 9 mutations, about 10mutations, about 11 mutations, about 12 mutations, about 13 mutations,about 14 mutations, about 15 mutations, about 16 mutations, about 17mutations, about 18 mutations, about 19 mutations, about 20 mutations,about 21 mutations, about 22 mutations, about 23 mutations, about 24mutations, about 25 mutations, about 26 mutations, about 27 mutations,about 28 mutations, about 29 mutations, about 30 mutations, about 31mutations, about 32 mutations, about 33 mutations, about 34 mutations,about 35 mutations, about 36 mutations, about 37 mutations, about 38mutations, about 39 mutations, about 40 mutations, about 41 mutations,about 42 mutations, about 43 mutations, about 44 mutations, about 45mutations, about 46 mutations, about 47 mutations, about 48 mutations,about 49 mutations, or about 50 mutations relative to the reference SHC(such as, for example, SEQ ID No. 1 or 2 or 3 or 4).

In these or other embodiments, the SHC/HAC derivative may comprise anamino acid sequence having at least about 50% sequence identity, atleast about 55% sequence identity, at least about 60% sequence identity,at least about 65% sequence identity, at least about 70% sequenceidentity, at least about 75% sequence identity, at least about 80%sequence identity, at least about 85% sequence identity, or at least 90%sequence identity, or at least 91% sequence identity, or at least 92%sequence identity, or at least 93% sequence identity, or at least 94%sequence identity, or at least 95% sequence identity, or at least 96%sequence identity, or at least 97% sequence identity, or at least 98%sequence identity, or at least 99% sequence identity, to WT SHC (suchas, for example, SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ IDNo. 4) or between the reference sequences (see for example Tables 18 and19 where at least 34-52% identity between AacSHC (SEQ ID No. 1) andother SHC sequences (eg. ZmoSHC of WO 2010/139719 is demonstrated).

In various embodiments, the SHC variant has higher activity forconverting homofarnesol to Ambrox than the wild-type enzyme, such as ahigher production of (−)-Ambrox upon contact with a homofarnesolsubstrate than the reference wild-type enzyme (such as, for example, SEQID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4).

For example, the SHC/HAC derivative may comprise an amino acid sequencehaving at least: about 50% identity, about 51% identity, about 52%identity, about 53% identity, about 54% identity, about 55% identity,about 56% identity, about 57% identity, about 58% identity, about 59%identity, about 60% identity, about 61% identity, about 62% identity,about 63% identity, about 64% identity, about 65% identity, about 66%identity, about 67% identity, about 68% identity, about 69% identity,about 70% identity, about 71% identity, about 72% identity, about 73%identity, about 74% identity, about 75% identity, about 76% identity,about 77% identity, about 78% identity, about 79% identity, about 80%identity, about 81% identity, about 82% identity, about 83% identity,about 84% identity, about 85% identity, about 86% identity, about 87%identity, about 88% identity, about 89% identity, about 90% identity,about 91% sequence identity, about 92% sequence identity, about 93%sequence identity, about 94% sequence identity, about 95% sequenceidentity, about 96% sequence identity, about 97% sequence identity,about 98% sequence identity, or about 99% sequence identity to thereference SHC (such as, for example SEQ ID No. 1 or 2 or 3 or 4) orbetween the reference sequences (see for example Tables 18 and 19 whereat least 34-52% identity between AacSHC (SEQ ID No. 1) and other SHCsequences (eg. ZmoSHC of WO 2010/139719) is demonstrated.

Various SHC/HAC derivatives which have been tested for SHC enzymeactivity are listed in one or more of Tables 1-9. Thus, in variousembodiments, the SHC/HAC derivative may have at least about 1, at leastabout 2, at least about 3, at least about 4, at least about 5, at leastabout 6, at least about 7, at least about 8, at least about 9, or atleast about 10 mutations selected from one or more of Tables 1-9. Insome embodiments, the SHC/HAC derivative is a modified SHC polypeptidecomprising an amino acid sequence which has up to 4 mutations comparedto the wild-type/reference amino acid sequence according to SEQ ID No. 1and comprises at least the substitutions F601Y or M132R in combinationwith at least any one or more of F129L and/or I432T relative to SEQ IDNo. 1 and optionally comprises a leader sequence supporting expressionand activity in E. coli.

In other embodiments, the SHC/HAC derivative is a modified SHCpolypeptide comprising an amino acid sequence which has up to 8mutations compared to the wild-type/reference amino acid sequenceaccording to SEQ ID No. 1 (or its counterpart that is modified forexpression in E. coli) and comprises one or more one amino acidalteration in a position selected from the group consisting of positions77, 92, 129, 132, 224, 432, 579, 601 and 605 relative to SEQ ID No. 1wherein the SHC/HAC derivative has an modified (eg. increased) enzymaticactivity relative to SEQ ID No. 1.

In one embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: T77X,192X, F129X, M132X, A224X, I432X, Q579X, F601Y, and F605W relative toSEQ ID No. 1 wherein:

T77X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

I92X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F129X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S T, V, W or Y.

M132X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

A224X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

I432X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N P, Q, R,S, T, V, W or Y.

Q579X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F601X has X selected from: A, C, D, E F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F605X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

In one embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: T77A,192V, F129L, M132R, A224V, I432T, Q579H, F601Y and F605W relative to SEQID No. 1.

In another embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: S129X,V145X, F182X, Y185X, G282X, I498X, H646X, and F698X relative to SEQ IDNo. 2 wherein:

S29X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

V145X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F182X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

Y185X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

G282X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

I498X has X selected from: A, B, C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W or Y.

H646X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F668X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T V, W or Y.

F698X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

In one embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: S129A,V145V, F182L, Y185R, G282V, I498T, H646H, F668Y and F698X relative toSEQ ID No. 2 as set out in Table 2.

In a further embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: G85X,V100X, F137X, I140X, V233X, I450X, N598X, F620X and F624X relative toSEQ ID No. 3 wherein:

G85X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

V100X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F137X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

I140X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

V233X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

I450X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

N598X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F620X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F624X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

In one embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: G85A,V100V, F137L, I140R, V233V, I450T, N598H, F620Y and F624W relative toSEQ ID No. 3 as set out in Table 3 and Table 3a.

In a further embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: A88X,V104X, F141X, Y144X, V241X, I459X, M607X, F628X and F658X relative toSEQ ID No. 4 wherein:

A88X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N P, Q, R, S,T, V, W or Y.

V104X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F141X, has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

Y144X has X selected from: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W or Y.

V241X has X selected from: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W or Y.

I459X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

M607X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F628X has X selected from: A, C, D, E, F, E, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

F658X has X selected from: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W or Y.

In a further embodiment, the SHC derivative comprises one or moresubstitutions selected from the group consisting of: A88A, V104V, F141L,Y144R, V241V, I459T, M607H, F628Y and F658W relative to SEQ ID No. 4 asset out in Table 4.

SHC Derivative Combinations

In one embodiment, the SHC derivative comprises one or moresubstitutions selected from the group of mutants consisting of: T77A,F129L, M132R, I92V, A224V, I432T, Q579H, and F601Y relative to SEQ IDNo. 1 as set out in Table 5.

In one embodiment, the SHC derivatives comprise one or moresubstitutions selected from the group of mutants consisting of: S129A,V145V F182L, Y185R, G282V, I498T, H646H and F668Y relative to SEQ ID No.2 as set out in Table 6.

In one embodiment, the SHC derivatives comprise one or moresubstitutions selected from the group of mutants consisting of: G85A,V100V F137L, I140R, V233V, I450T, N598H, and F620Y relative to SEQ IDNo. 3 as set out in Table 7.

In a further embodiment, the SHC derivative comprises one or moresubstitutions selected from the group consisting of: A88A, V104V, F141L,Y144R, V241V, I459T, M607H, and F628Y relative to SEQ ID No. 4 as setout in Table 8.

TABLE 1 Summary of SHC mutations numbered relative to wild-type AacSHC(SEQ ID No. 1) and FIG. 1 from Hoshino and Sato (2002 as cited above)Amino Acid Amino Acid (H. sapiens) Amino Acid Position Amino Acid(ZmoSHC) (Hoshino in in WT (AacSHC) (Hoshino and and Sato SEQ ID No. No.AacSHC Location (SEQ ID No. 1) Sato 2002) 2002) Mutation (Table 14) 1 77QW5c-QW5b T G G A 5 8 92 QW5c-QW5b I V A V 7 2 129 QW5c-QW5b F F W L 9 3132 QW5c-QW5b M I F R 11 4 224 QW5c-QW5b A V V V 13 5 432 QW4-QW3 I I NT 15 6 579 QW1 Q N R H 17 7 601 QW1 F F F Y 19 9 605 F F F W 171

TABLE 2 Summary of SHC mutations numbered relative to wild-type AacSHC(SEQ ID No. 1) and the sequence alignment of AacSHC with ZmoSHC1 (Seitzet al 2012 - as cited above) Supplementary Data Sheet PositionEquivalent and AA in AA and WT AacSHC position in AA in (SEQ ID ZmoSHC1SEQ ID No. No. No. 1) (SEQ ID No. 2) Mutation (see Table 15) 1 77 T S129 A 41 8 92 I V 145 V 43 2 129 F F 182 L 45 3 132 M Y 185 R 47 4 224 AG 282 V 49 5 432 I I 498 T 51 6 579 Q H 647 H 53 7 601 F F 668 Y 55 9605 F F 698 W 173

TABLE 3 Summary of SHC mutations numbered relative to wild-type AacSHC(SEQ ID No. 1) and the sequence alignment of AacSHC with ZmoSHC2 (Seitzet al 2012 - as cited above) Supplementary Data Sheet Position AA andand AA in position in AA in SEQ ID WT AacSHC ZmoSHC2 No. No. (SEQ IDNo. 1) (SEQ ID No. 3) Mutation (see Table 16) 1 77 T G 85 A 77 8 92 I V100 V 79 2 129 F F 137 L 81 3 132 M I 140 R 83 4 224 A V 233 V 85 5 432I I 450 T 87 6 579 Q N 598 H 89 7 601 F F 620 Y 91 9 605 F F 624 W 175

TABLE 3a Summary of SHC mutations numbered relative to wild-type AacSHC(SEQ ID No. 1) and the sequence alignment of AacSHC with sequence No. 20in the SHC Alignment Figure of Merkofer PhD thesis (2004) (seehttp://elib.uni-stuttgart.de/handle/11682/1400) Position and AA in WTAacSHC AA in No. (SEQ ID No. 1) ZmoSHC Mutation 1 77 T G A 8 92 I V V 2129 F F L 3 132 M I R 4 224 A V V 5 432 I I T 6 579 Q N H 7 601 F F Y 9605 F F W

TABLE 4 Summary of SHC mutations numbered relative to wild-type AacSHC(SEQ ID No. 1) and the sequence alignment of AacSHC with BjpSHC (SEQ IDNo. 5 in WO 2010/139719) Position AA and and AA in position in AA in WTAacSHC WT BjpSHC SEQ ID No. No. (SEQ ID No. 1) (SEQ ID No. 4) Mutation(See Table 17) 1 77 T A 88 A 113 8 92 I V 104 V 115 2 129 F F 141 L 1173 132 M Y 144 R 119 4 224 A V 241 V 121 5 432 I I 459 T 123 6 579 Q M607 H 125 7 601 F F 628 Y 127 9 605 F F 658 W 177

TABLE 4a Using the sequence alignments provided in Tables 21a-21j whereWTAacSHC (SEQ ID No. 1) is aligned with any one of SEQ ID No. 149, 151,153, 155, 157, 159 (as identified below), the AA residue and positionscorresponding to T77, I92, F129, M132, A224, I432, Q579 and F601 in WTAacSHC (SEQ ID No. 1) can be identified and tested for SHC/HAC activitySEQ ID No. Nucleo- in WO 2010/ tide/ 0139719 amino (nucleotide/ acidAmino Acid amino acid SEQ SEQ ID No. Strain sequences) ID No. SEQ ID No.149 Burkholderia ambifaria6 SEQ ID No. 6 150 SEQ ID No. 151 Burkholderiaambifaria SEQ ID No. 7 152 SEQ ID No. 153 Bacillus anthracis SEQ ID No.8 154 SEQ ID No. 155 Frankia alni SEQ ID No. 9 156 SEQ ID No. 157Rhodopseudomonas palent  SEQ ID No. 10 158 SEQ ID No. 159 Streptomycescoelicolor  SEQ ID No. 11 160 SEQ ID No. 161 Zymomonas mobilis2 SEQ IDNo. 2 162 SEQ ID No. 163 Zymomonas mobilis SEQ ID No. 1 164 SEQ ID No.4  Bradyrhizobium japonicum5 SEQ ID No. 5 168

TABLE 5 Summary of SHC mutations combinations numbered according towild-type AacSHC (SEQ ID No. 1) Amino Nucleo- Mutation combinations SHCNumber acid tide in AacSHC Derivative of SEQ ID SEQ ID (Seq ID No. 1) IDmutations No. No. M132R + A224V + I432T 215G2 3 21 22 M132R + I432TSHC26 2 23 24 F601Y SHC3 1 25 26 T77A + I92V + F29L 111C8 3 27 28Q579H + F601Y 101A10 2 29 30 F129L SHC10 1 31 32 F29L + F601Y SHC30 2 3334 F29L + M132R + I432T SHC31 3 35 36 M132R + I432T + F601Y SHC32 3 3738 F129L + M132R + I432T + SHC33 4 39 40 F6601Y

TABLE 6 Summary of SHC mutations combinations numbered according towild-type ZmoSHC1 sequence (SEQ. ID No. 2) Amino Nucleo- Mutationcombinations SHC Number acid tide in ZmoSHC1 Derivative of SEQ SEQ (SEQID No. 2) ID mutations ID No. ID No. Y185R + G282V + I498T 215G2ZM1 3 5758 Y185R + I498T SHC26 ZM1 2 59 60 F668Y SHC3 ZM1 1 61 62 S129A +V145V + F182L 111C8 ZM1 3 63 64 H646H + F668Y 101A10 ZM1 7 65 66 F182LSHC10 ZM1 1 67 68 F182L + F668Y SHC30 ZM1 2 69 70 F182L + Y185R + I498TSHC31 ZM1 3 71 72 Y185R + I498T + F668Y SHC32 ZM1 3 73 74 F182L +Y185R + I498T + SHC33 ZM1 4 75 76 F668Y

TABLE 7 Summary of SHC n utations combinations numbered according towild-type ZmoSHC2 sequence (SEQ. ID No. 3) Amino Nucleo- Mutationcombinations SHC Number acid tide in ZmoSHC2 Derivative of SEQ SEQ (SEQID No. 3) ID mutations ID No. ID No. I140R + V233V + I450T 215G2 ZM2 393 94 I140R + I450T SHC26 ZM2 2 95 96 F620Y SHC3 ZM2 1 97 98 G85A +V100V + F137L 111C8 ZM2 3 99 100 N598H + F620Y 101A10 ZM2 2 101 102F137L SHC10 ZM2 1 103 104 F137L + F620Y SHC30 ZM2 2 105 106 F137L +I140R + I450T SHC31 ZM2 3 107 108 I140R + I450T + F620Y SHC32 ZM2 3 109110 F137L + I140R + SHC33 ZM2 4 111 112 I450T + F620Y

TABLE 8 Summary of SHC mutations combinations numbered according towild-type BjpSHC (SEQ ID No. 4) Amino Nucleo- Mutation combinations SHCNumber acid tide in BjpSHC Derivative of SEQ SEQ (SEQ ID No. 4) IDmutations ID No. ID No. Y144R + V241V + I459T 215G2 Bjp 3 129 130V144R + I459T SHC26 BjP 2 131 132 F628Y SHC3 Bjp 1 133 134 A88A +V104V + F141L 111C8 Bjp 3 135 136 M607H + F628Y 101A10 Bjp 2 137 138F141L SHC10 Bjp 1 139 140 F141L + F628Y SHC30 Bjp 2 141 142 F141L +Y144R + I459T SHC31 Bjp 3 143 144 M144R + I459T + F628Y SHC32 Bjp 3 145146 F14IL + Y144R + SHC33 Bjp 4 147 148 I459T + F628Y

TABLE 9 Showing the common SHC mutations relative to the WT AacSHC (SEQID No. 1), WT ZmoSHC1 (SEQ ID No. 2), WT ZmoSHC2 (SEQ ID No. 3) andBjpSHC (SEQ ID No. 4) Mutation Combinations Relative to Relative toRelative to in AacSHC ZmoSHC1 ZmoSHC2 BjpSHC SHC (SEQ ID No. 1) (SEQ IDNo. 2) (SEQ ID No. 3) (SEQ ID No. 4) Derivative F601Y F668Y F620Y F628YSHC3  F129L F182L F137L F141L SHC10 F601Y + F129L  F668Y + F182L F620Y + F137L  F628Y + F141L  SHC30 F601Y + M132R + F668Y + Y185R +F620Y + I140R + F628Y + Y144R + SHC32 I432T I498T I450T I459T F601Y +F129L + F668Y + F182L + F620Y + F137L + F628Y + F141L + SHC33 M132R +I432T  Y185R + I498T  I140R + I450T  Y144R + I459T  M132R + I432T Y185R + I498T  I140R + I450T  Y144R + I459T  SHC26 M132R + I432T +Y185R + I498T + I140R + I450T + Y144R + I459T + 215G2 A224V G282V V233VV241V M132R + I432T + Y185R + I498T + I140R + I450T + Y144R + I459T +SHC31 F129L F182L F137L F141L

In a preferred embodiment, the SHC derivative comprises at least thesubstitutions F601Y or M132R in combination with at least any one ormore of F129L and/or I432T relative to SEQ ID No. 1.

The SHC derivative termed SHC3 which is provided in the presentdisclosure comprises the following substitution F601Y as compared withthe reference SHC protein (SEQ ID No. 1).

Hoshino and Sato (2002 as cited above) identified F601 as a highlyconserved amino acid residue among the prokaryotic and eukaryoticspecies. It is reported that SHC derivative F601Y showed a greatlyincreased Vmax for an oxidosqualene substrate (not squalene). HoweverF601Y shows a decrease in affinity (i.e. a higher K_(M)) and a decreasein catalytic efficiency/activity (Kcat/K_(M)) relative to the WT AacSHCwhen squalene is used. No data is provided in Hoshino and Sato (2002 ascited above) on AacSHC efficacy when homofarnesol is used as the enzymesubstrate with the F601Y mutant.

The SHC derivative termed SHC10 which is provided in the presentdisclosure comprises the following substitution F129L as compared withthe reference SHC protein (SEQ ID No. 1).

The SHC derivative termed SHC30 which is provided in the presentdisclosure comprises the following substitution F601Y and F129L ascompared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed SHC26 which is provided in the presentdisclosure comprises the following substitution M132R and I432T ascompared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed 215G2 which is provided in the presentdisclosure comprises the following substitution M132R, I432T and A224Vas compared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed SHC32 which is provided in the presentdisclosure comprises the following substitution F601Y, M132R and I432Tas compared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed SHC31 which is provided in the presentdisclosure comprises the following substitution F129L, M132R and I432Tas compared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed SHC33 which is provided in the presentdisclosure comprises the following substitution F601Y, F129L, M132R andI432T as compared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed 101A10 which is provided in the presentdisclosure comprises the following substitution F601Y and Q579H ascompared with the reference SHC protein (SEQ ID No. 1).

The SHC derivative termed 111C8 which is provided in the presentdisclosure comprises the following substitution T77A+I92V and F129L ascompared with the reference SHC protein (SEQ ID No. 1).

In a preferred embodiment, the SHC derivative comprises at least thesubstitutions F668Y or Y185R in combination with at least any one ormore of F182L and/or I498T relative to SEQ ID No. 2.

The SHC derivative termed SHC3ZM1 which is provided in the presentdisclosure comprises the following substitution F668Y as compared withthe reference SHC protein (SEQ ID No. 2).

Hoshino and Sato (2002 as cited above) identified F601 as a highlyconserved amino acid residue among the prokaryotic and eukaryoticspecies. It is reported that SHC derivative F601Y showed a greatlyincreased Vmax for a oxidosqualene substrate (not squalene). HoweverF601Y shows a decrease in affinity (i.e. a higher K_(M)) and a decreasein catalytic efficiency/activity (Kcat/K_(M)) relative to the WT AacSHCwhen squalene is used. No data is provided in Hoshino and Sato on AacSHCefficacy when Homofarnesol is used as the enzyme substrate with theF601Y mutant. The SHC derivative equivalent to F601Y in ZmoSHC1 isF668Y.

The SHC derivative termed SHC10ZM1 which is provided in the presentdisclosure comprises the following substitution F182L as compared withthe reference SHC protein (SEQ ID No. 2).

The SHC derivative termed SHC30ZM1 which is provided in the presentdisclosure comprises the following substitution F668Y and F182L ascompared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative teemed SHC26ZM1 which is provided in the presentdisclosure comprises the following substitution Y185R and I498T ascompared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative termed 215G2ZM1 which is provided in the presentdisclosure comprises the following substitution Y185R, I498T and G282Vas compared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative termed SHC32ZM1 which is provided in the presentdisclosure comprises the following substitution F668Y, Y185R and I498Tas compared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative termed SHC31ZM1 which is provided in the presentdisclosure comprises the following substitution F182L, Y185R and I498Tas compared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative termed SHC33ZM1 which is provided in the presentdisclosure comprises the following substitution F668Y, F182L, Y185R andI498T as compared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative termed I01A10ZM1 which is provided in the presentdisclosure comprises the following substitution F668Y and H646H ascompared with the reference SHC protein (SEQ ID No. 2).

The SHC derivative termed 111C8ZM1 which is provided in the presentdisclosure comprises the following substitution S129A+V145V and F182L ascompared with the reference SHC protein (SEQ ID No. 2).

In a preferred embodiment, the SHC derivative comprises at least thesubstitutions F620Y or I140R in combination with at least any one ormore of F137L and/or I450T relative to SEQ ID No. 3.

The SHC derivative termed SHC3ZM2 which is provided in the presentdisclosure comprises the following substitution F620Y as compared withthe reference SHC protein (SEQ ID No. 3).

Hoshino and Sato (2002 as cited above) identified F601 as a highlyconserved amino acid residue among the prokaryotic and eukaryotic SHCspecies. It was reported that AacSHC derivative F601Y showed a greatlyincreased Vmax for a oxidosqualene substrate (not squalene). HoweverF601Y shows a decrease in affinity (i.e. a higher K_(M)) and a decreasein catalytic efficiency/activity (Kcat/K_(M)) relative to the WT AacSHCwhen squalene is used. No data is provided in Hoshino and Sato (2002) onAacSHC efficacy when Homofarnesol is used as the enzyme substrate withthe F601Y mutant. The SHC derivative equivalent to F601Y in ZmoSHC2 isF620Y.

The SHC derivative termed SHC10ZM2 which is provided in the presentdisclosure comprises the following substitution F137L as compared withthe reference SHC protein (SEQ ID No. 3).

The SHC derivative termed SHC30ZM2 which is provided in the presentdisclosure comprises the following substitution F620Y and F137L ascompared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed SHC26ZM2 which is provided in the presentdisclosure comprises the following substitution I140R and I450T ascompared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed 215G2ZM2 which is provided in the presentdisclosure comprises the following substitution I140R, I450T and V233Vas compared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed SHC32ZM2 which is provided in the presentdisclosure comprises the following substitution F620Y, I140R and I450Tas compared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed SHC3 ZM2 which is provided in the presentdisclosure comprises the following substitution F137L, I140R and I450Tas compared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed SHC33 ZM2 which is provided in the presentdisclosure comprises the following substitution F620Y, F137L, I140R andI450T as compared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed 101A10ZM2 which is provided in the presentdisclosure comprises the following substitution F620Y and N598H ascompared with the reference SHC protein (SEQ ID No. 3).

The SHC derivative termed 111C8ZM2 which is provided in the presentdisclosure comprises the following substitution G85A+V100V and F137L ascompared with the reference SHC protein (SEQ ID No. 3).

In a preferred embodiment, the SHC derivative comprises at least thesubstitutions F628Y or Y144R in combination with at least any one ormore of F141L and/or I459T relative to SEQ ID No. 4.

The SHC derivative termed SHC3Bjp which is provided in the presentdisclosure comprises the following substitution F628Y as compared withthe reference SHC protein (SEQ ID No. 4).

Hoshino and Sato (2002 as cited above) identified F601 as a highlyconserved amino acid residue among the prokaryotic and eukaryoticspecies. It is reported that SHC derivative F601 Y showed a greatlyincreased Vmax for a oxidosqualene substrate (not squalene). HoweverF601Y shows a decrease in affinity (i.e. a higher K_(M)) and a decreasein catalytic efficiency/activity (Kcat/K_(M)) relative to the WT AacSHCwhen squalene is used. No data is provided in Hoshino and Sato on AacSHCefficacy when Homofarnesol is used as the enzyme substrate with theF601Y mutant. The SHC derivative equivalent to F601Y in BjpSHC is F628Y.

The SHC derivative termed SHC10Bjp which is provided in the presentdisclosure comprises the following substitution F141L as compared withthe reference SHC protein (SEQ ID No. 4).

The SHC derivative termed SHC30Bjp which is provided in the presentdisclosure comprises the following substitution F628Y and F141L ascompared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed SHC26Bjp which is provided in the presentdisclosure comprises the following substitution Y144R and I459T ascompared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed 215G2Bjp which is provided in the presentdisclosure comprises the following substitution Y144R, I459T and V241Vas compared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed SHC32Bjp which is provided in the presentdisclosure comprises the following substitution F628Y, Y144R and I459Tas compared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed SHC31Bjp which is provided in the presentdisclosure comprises the following substitution F141L, Y144R and I459Tas compared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed SHC33Bjp which is provided in the presentdisclosure comprises the following substitution F628Y, F141L, Y144R andI459T as compared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed 101A10Bjp which is provided in the presentdisclosure comprises the following substitution F628Y and M607H ascompared with the reference SHC protein (SEQ ID No. 4).

The SHC derivative termed 111C8Bjp which is provided in the presentdisclosure comprises the following substitution A88A+V104V and F141L ascompared with the reference SHC protein (SEQ ID NO: 4).

Amino Acid Sequences

In some embodiments, the AacSHC/HAC derivative comprises one or more ofthe polypeptides as set out in one or more of SEQ ID No. 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and/or 171.

Preferably the AacSHC/HAC derivatives of the present disclosure have anamino acid sequence selected from the group consisting of SEQ ID No. 21,SEQ ID No. 23, SEQ ID No. 25 SEQ ID No. 27, SEQ ID No. 29, SEQ ID No.31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39 and/orSEQ ID No. 171.

In other embodiments, the ZmoSHC1/HAC derivatives comprise one or moreof the polypeptides as set out in one or more of SEQ ID No. 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 and/or 173.

Preferably the ZmoSHC1/HAC derivatives of the present disclosure have anamino acid sequence selected from the group consisting of SEQ ID No. 57,SEQ ID No. 59, SEQ ID No. 61, SEQ ID No. 63, SEQ ID No. 65, SEQ ID No.67, SEQ ID No. 69, SEQ ID No. 71, SEQ ID No. 73, SEQ ID No. 75 and/orSEQ ID No. 173.

In further embodiments, the ZmoSHC2/HAC derivatives comprise one or moreof the polypeptides as set out in one or more of SEQ ID No. 77, SEQ IDNo. 79, SEQ ID No. 81, SEQ ID No. 83, SEQ ID No. 85, SEQ ID No. 87, SEQID No. 89, SEQ ID No. 91, SEQ ID No. 93, SEQ ID No. 95, SEQ ID No. 97,SEQ ID No. 99, SEQ ID No. 101, SEQ ID No. 103, SEQ ID No. 105, SEQ IDNo. 107, SEQ ID No. 109, SEQ ID No. 111 and/or SEQ ID No. 175.

In additional embodiments, the BjpSHC/HAC derivatives comprise one ormore of the polypeptides as set out in one or more of: SEQ ID No. 113,SEQ ID No. 115, SEQ ID No. 117, SEQ ID No. 119, SEQ ID No. 121, SEQ IDNo. 123, SEQ ID No. 125, SEQ ID No. 127, SEQ ID No. 129, SEQ ID No. 131.SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 137, SEQ ID No. 139, SEQ IDNo. 141, SEQ ID No. 143, SEQ ID No. 145, SEQ ID No. 147 and/or SEQ IDNo. 177.

Sequence Alignments

Due to the different lengths of SHC reference sequences, such as, forexample, the AacSHC, ZmoSHC1, ZmoSHC2 and BjpSHC polypeptides sequences,the amino acid residue at position X of the reference AacSHC sequence(SEQ ID No. 1) corresponds to a different amino acid position B on theZmoSHC1 reference sequence (SEQ ID No. 2), a different amino acidposition J on the ZmoSHC2 reference sequence (SEQ ID No. 3) and adifferent amino acid position Z on the BjpSHC reference sequence (SEQ IDNo. 4), In addition, the alteration of an SHC reference sequence canalso modify the SHC derivative sequence relative that reference SHCsequence.

The term “position” refers to a specific amino acid residue present inthe reference SHC protein as identified by the specific numbering of theamino acids. The alteration of the SHC reference protein by either aninsertion or a deletion of an amino acid leads to a different numberingbetween the reference SHC amino acid sequence and the SHC derivativeamino acid sequence. By way of example, if an amino acid is insertedbetween amino acids 509 and 510 of the reference SHC protein, the aminoacid following the insertion will have the numbering 511 in the SHCderivative protein while it retains the numbering 510 in the SHCreference protein.

Assays for Determining WT SHC/HAC and SHC/HAC Derivative Activity

Assays for determining and quantifying WT SHC/HAC and/or SHC/HACderivative enzyme activity are described herein and are known in theart. By way of example, WT SHC/HAC and/or SHC/HAC derivative activitycan be determined by incubating purified SHC/HAC enzyme or extracts fromhost cells or a complete recombinant host organism that has produced theSHC/HAC enzyme with an appropriate substrate under appropriateconditions and carrying out an analysis of the reaction products (eg. bygas chromatography (GC) or HPLC analysis). Further details on SHC/HACand/or SHC/HAC enzyme activity assays and analysis of the reactionproducts are provided in the Examples. These assays include producingthe SHC derivative in recombinant host cells (eg. E. coli).

As used herein, the term “activity” means the ability of an enzyme toreact with a substrate to provide a target product. The activity can bedetermined in what is known as an activity test via the increase of thetarget product, the decrease of the substrate (or starting materials) orvia a combination of these parameters as a function of time. The SHC/HACderivatives of the present disclosure are characterized by their abilityto bioconvert homofarnesol into (−)-Ambrox and demonstrate a biologicalactivity such as an HAC activity.

A “biological activity” as used herein, refers to any activity apolypeptide may exhibit, including without limitation: enzymaticactivity; binding activity to another compound (eg. binding to anotherpolypeptide, in particular binding to a receptor, or binding to anucleic acid); inhibitory activity (eg. enzyme inhibitory activity);activating activity (eg. enzyme-activating activity); or toxic effects.It is not required that the variant or derivative exhibits such anactivity to the same extent as the parent polypeptide. A variant isregarded as a variant within the context of the present application, ifit exhibits the relevant activity to a degree of at least 10% of theactivity of the parent polypeptide. Likewise, a derivative is regardedas a derivative within the context of the present application, if itexhibits the relevant biological activity to a degree of at least 10% ofthe activity of the parent polypeptide (as the terms derivative andvariant are used interchangeably throughout the present disclosure).

In other embodiments, the SHC/HAC derivatives of the present disclosureshow a better target yield than the reference SHC protein. The term“target yield” refers to the gram of recoverable product per grain offeedstock (which can be calculated as a percent molar conversion rate).

In additional embodiments, the SHC/HAC derivatives of the presentdisclosure show a modified (eg. increased) target productivity relativeto the reference SHC protein. The term “target productivity” refers tothe amount of recoverable target product in grams per liter offermentation capacity per hour of bioconversion time (i.e. time afterthe substrate was added).

In further embodiments, the SHC/HAC derivatives of the presentdisclosure show a modified target yield factor than the reference SHCprotein. The term “target yield factor” refers to the ratio between theproduct concentration obtained and the concentration of the SHCderivative (for example, purified SHC enzyme or an extract from therecombinant host cells expressing the SHC enzyme) in the reactionmedium.

In various embodiments, the SHC derivatives of the present disclosureshow a modified (eg. increased) fold increase in enzymatic activity (eg.a modified/increased homofarnesol Ambrox cyclase (HAC) activity)relative to the reference SHC protein (eg. SEQ ID No. 1 or SEQ ID No. 2or SEQ ID No. 3 or SEQ ID No. 4). This increase in activity is at leastby a factor of: 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and/or 100.

Nucleotide Sequences

The present disclosure further relates to isolated nucleic acidmolecules comprising a nucleotide sequence encoding an SHC derivative asdescribed herein.

The term “nucleic acid molecule” as used herein shall specifically referto polynucleotides of the disclosure which can be DNA, cDNA, genomicDNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded, the sense and/or an antisense strand. The term “nucleicacid molecule” shall particularly apply to the polynucleotide(s) as usedherein, eg. as full-length nucleotide sequence or fragments or partsthereof, which encodes a polypeptide with enzymatic activity, eg. anenzyme of a metabolic pathway, or fragments or parts thereof,respectively.

The term also includes a separate molecule such as a cDNA where thecorresponding genomic DNA has introns and therefore a differentsequence; a genomic fragment that lacks at least one of the flankinggenes; a fragment of cDNA or genomic DNA produced by polymerase chainreaction (PCR) and that lacks at least one of the flanking genes; arestriction fragment that lacks at least one of the flanking genes; aDNA encoding a non-naturally occurring protein such as a fusion protein(eg. a His tag), mutein, or fragment of a given protein; and a nucleicacid which is a degenerate variant of a cDNA or a naturally occurringnucleic acid. In addition, it includes a recombinant nucleotide sequencethat is part of a hybrid gene, i.e. a gene encoding a non-naturallyoccurring fusion protein. Fusion proteins can add one or more aminoacids (such as but not limited to Histidine (His)) to a protein, usuallyat the N-terminus of the protein but also at the C-terminus or fusedwithin regions of the protein. Such fusion proteins or fusion vectorsencoding such proteins typically serve three purposes: (i) to increaseproduction of recombinant proteins; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification of therecombinant protein by providing a ligand for affinity purification. Theterm “nucleic acid molecule” also includes codon optimised sequencessuitable for expression in a particular microbial host cell (eg. E. colihost cell). As used herein, the term “codon optimized” means a nucleicacid protein coding sequence which has been adapted for expression in aprokaryotic or a eukaryotic host cell, particularly bacterial host cellssuch as E. coli host cells by substitution of one or more or preferablya significant number of codons with codons that are more frequently usedin bacterial (eg. E. coli) host cell genes. In this regard, thenucleotide sequence encoding the reference sequences Sequence ID No. 1,2, 3 and/or 4 and all variants/derivatives thereof may be the originalone as found in the source (eg. AacSHC, ZmoSHC1, ZmoSHC2 or BjpSHCrespectively) or the gene can be codon-optimized for the selected hostorganisms, such as eg. E. coli.

A ribonucleic acid (RNA) molecule can be produced by in vitrotranscription. Segments of DNA molecules are also considered within thescope of the disclosure, and can be produced by, for example, thepolymerase chain reaction (PCR) or generated by treatment with one ormore restriction endonucleases. Segments of a nucleic acid molecule maybe referred to as DNA fragments of a gene, in particular those that arepartial genes. A fragment can also contain several open reading frames(ORF), either repeats of the same ORF or different ORF's. The term shallspecifically refer to coding nucleotide sequences, but shall alsoinclude nucleotide sequences which are non-coding, eg. untranscribed oruntranslated sequences, or encoding polypeptides, in whole or in part.The genes as used herein, eg. for assembly, diversification orrecombination can be non-coding sequences or sequences encodingpolypeptides or protein encoding sequences or parts or fragments thereofhaving sufficient sequence length for successful recombination events.More specifically, said genes have a minimum length of 3 bp, preferablyat least 100 bp, more preferred at least 300 bp.

It will be apparent from the foregoing that a reference to n isolatedDNA does not mean a DNA present among hundreds to millions of other DNAmolecules within, for example, cDNA or genomic DNA libraries or genomicDNA restriction digests in, for example, a restriction digest reactionmixture or an electrophoretic gel slice. An isolated nucleic acidmolecule of the present disclosure encompasses segments that are notfound as such in the natural state.

As used herein, the term “isolated DNA” can refer to (1) a DNA thatcontains sequence not identical to that of any naturally occurringsequence, a polynucleotide or nucleic acid which is not naturallyoccurring, (eg., is made by the artificial combination (eg. artificialmanipulation of isolated segments of nucleic acids, eg., by geneticengineering techniques) of two otherwise separated segments of sequencesthrough human intervention) or (2), in the context of a DNA with anaturally-occurring sequence (eg., a cDNA or genomic DNA), a DNA free ofat least one of the genes that flank the gene containing the DNA ofinterest in the genome of the organism in which the gene containing theDNA of interest naturally occurs.

The term “isolated DNA” as used herein, specifically with respect tonucleic acid sequence may also refer to nucleic acids or polynucleotidesproduced by recombinant DNA techniques, eg. a DNA construct comprising apolynucleotide heterologous to a host cell, which is optionallyincorporated into the host cell. A chimeric nucleotide sequence mayspecifically be produced as a recombinant molecule. The term“recombination” shall specifically apply to assembly of polynucleotides,joining together such polynucleotides or parts thereof, with or withoutrecombination to achieve a cross-over or a gene mosaic. For example, itis performed to join together nucleic acid segments of desired functionsto generate a desired combination of functions. A recombinant geneencoding a polypeptide described herein includes the coding sequence forthat polypeptide, operably linked, in sense orientation, to one or moreregulatory regions suitable for expressing the polypeptide. Because manymicroorganisms are capable of expressing multiple gene products from apolycistronic mRNA, multiple polypeptides can be expressed under thecontrol of a single regulatory region for those microorganisms, ifdesired. A coding sequence and a regulatory region are considered to beoperably linked when the regulatory region and coding sequence arepositioned so that the regulatory region is effective for regulatingtranscription or translation of the sequence.

The term “recombinant” as used herein, specifically with respect toenzymes shall refer to enzymes produced by recombinant DNA techniques,i.e. produced from cells transformed by an exogenous DNA constructencoding the desired enzyme. “Synthetic” enzymes are those prepared bychemical synthesis. A chimeric enzyme may specifically be produced asrecombinant molecule. The term “recombinant DNA” therefore includes arecombinant DNA incorporated into a vector into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote (or the genome of a homologous cell, at a position other thanthe natural chromosomal location).

In a further aspect the nucleic acid molecule(s) of the presentdisclosure is/are operatively linked to expression control sequencesallowing expression in prokaryotic and/or eukaryotic host cells. As usedherein, “operatively linked” means incorporated into a genetic constructso that expression control sequences effectively control expression of acoding sequence of interest. The transcriptional/translationalregulatory elements referred to above include but are not limited toinducible and non-inducible, constitutive, cell cycle regulated,metabolically regulated promoters, enhancers, operators, silencers,repressors and other elements that are known to those skilled in the artand that drive or otherwise regulate gene expression. Such regulatoryelements include but are not limited to regulatory elements directingconstitutive expression or which allow inducible expression like, forexample, CUP-1 promoter, the tet-repressor as employed, for example, inthe tet-on or tet-off systems, the lac system, the trp system regulatoryelements. By way of example, Isopropyl β-D-1-thiogalactopyranoside(IPTG) is an effective inducer of gene expression in the concentrationrange of 100 μM to 1.0 mM. This compound is a molecular mimic ofallolactose, a lactose metabolite that triggers transcription of the lacoperon, and it is therefore used to induce gene expression when the geneis under the control of the lac operator. Another example of aregulatory element which induces gene expression is lactose.

Similarly, the nucleic acid molecule(s) of the present disclosure canform part of a hybrid gene encoding additional polypeptide sequences,for example, a sequence that functions as a marker or reporter. Examplesof marker and reporter genes including beta-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ(encoding beta-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the disclosure, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter.

In some embodiment, the present disclosure provides a recombinantpolynucleotide encoding the WT SHC or the SHC/HAC derivative describedabove, which may be inserted into a vector for expression and optionalpurification. One type of vector is a plasmid representing a circulardouble stranded DNA loop into which additional DNA segments are ligated.Certain vectors can control the expression of genes to which they arefunctionally linked. These vectors are called “expression vectors”.Usually expression vectors suitable for DNA recombination techniques areof the plasmid type. Typically, an expression vector comprises a genesuch as the WT SHC or the SHC/HAC variant as described herein. In thepresent description, the terms “plasmid” and “vector” are usedinterchangeably since the plasmid is the vector type most often used.

Such vectors can include DNA sequences which include but are not limitedto DNA sequences that are not naturally present in the host cell, DNAsequences that are not normally transcribed into RNA or translated intoa protein (“expressed”) and other genes or DNA sequences which onedesires to introduce into the non-recombinant host. It will beappreciated that typically the genome of a recombinant host describedherein is augmented through the stable introduction of one or morerecombinant genes. However, autonomous or replicative plasmids orvectors can also be used within the scope of this disclosure. Moreover,the present disclosure can be practiced using a low copy number, eg., asingle copy, or high copy number (as exemplified herein) plasmid orvector.

In a preferred embodiment the vector of the present disclosure comprisesplasmids, phagemids, phages, cosmids, artificial bacterial andartificial yeast chromosomes, knock-out or knock-in constructs,synthetic nucleic acid sequences or cassettes and subsets may beproduced in the form of linear polynucleotides, plasmids, megaplasmids,synthetic or artificial chromosomes, such as plant, bacterial, mammalianor yeast artificial chromosomes.

It is preferred that the proteins encoded by the introducedpolynucleotide are expressed within the cell upon introduction of thevector. The diverse gene substrates may be incorporated into plasmids.The plasmids are often standard cloning vectors, eg., bacterialmulticopy plasmids. The substrates can be incorporated into the same ordifferent plasmids. Often at least two different types of plasmid havingdifferent types of selectable markers are used to allow selection forcells containing at least two types of vectors.

Typically bacterial or yeast cells may be transformed with any one ormore of the following nucleotide sequences as is well known in the art.For in vivo recombination, the gene to be recombined with the genome orother genes is used to transform the host using standard transformingtechniques. In a suitable embodiment DNA providing an origin ofreplication is included in the construct. The origin of replication maybe suitably selected by the skilled person. Depending on the nature ofthe genes, a supplemental origin of replication may not be required ifsequences are already present with the genes or genome that are operableas origins of replication themselves.

A bacterial or yeast cell may be transformed by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated, i.e. covalently linkedinto the genome of the cell. In prokaryotes, and yeast, for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transfected cell isone in which the transfected DNA has become integrated into a chromosomeso that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transforming DNA.

Generally, the introduced DNA is not originally resident in the hostthat is the recipient of the DNA, but it is within the scope of thedisclosure to isolate a DNA segment from a given host, and tosubsequently introduce one or more additional copies of that DNA intothe same host, eg., to enhance production of the product of a gene oralter the expression pattern of a gene. In some instances, theintroduced DNA will modify or even replace an endogenous gene or DNAsequence by, eg., homologous recombination or site-directed mutagenesis.Suitable recombinant hosts include microorganisms, plant cells, andplants.

The present disclosure also features recombinant hosts. The term“recombinant host”, also referred to as a “genetically modified hostcell” or a “transgenic cell” denotes a host cell that comprises aheterologous nucleic acid or the genome of which has been augmented byat least one incorporated DNA sequence. A host cell of the presentdisclosure may be genetically engineered with the polynucleotide or thevector as outlined above.

The host cells that may be used for purposes of the disclosure includebut are not limited to prokaryotic cells such as bacteria (for example,E. coli and B. subtilis), which can be transformed with, for example,recombinant bacteriophage DNA, plasmid DNA, bacterial artificialchromosome, or cosmid DNA expression vectors containing thepolynucleotide molecules of the disclosure; simple eukaryotic cells likeyeast (for example, Saccharomyces and Pichia), which can be transformedwith, for example, recombinant yeast expression vectors containing thepolynucleotide molecule of the disclosure. Depending on the host celland the respective vector used to introduce the polynucleotide of thedisclosure the polynucleotide can integrate, for example, into thechromosome or the mitochondrial DNA or can be maintainedextrachromosomally like, for example, episomally or can be onlytransiently comprised in the cells.

The term “cell” as used herein in particular with reference to geneticengineering and introducing one or more genes or an assembled cluster ofgenes into a cell, or a production cell is understood to refer to anyprokaryotic or eukaryotic cell. Prokaryotic and eukaryotic host cellsare both contemplated for use according to the disclosure, includingbacterial host cells like E. coli or Bacillus sp, yeast host cells, suchas S. cerevisiae, insect host cells, such as Spodoptera frugiperda orhuman host cells, such as HeLa and Jurkat.

Specifically, the cell is a eukaryotic cell, preferably a fungal,mammalian or plant cell, or prokaryotic cell. Suitable eucaryotic cellsinclude, for example, without limitation, mammalian cells, yeast cells,or insect cells (including Sf9), amphibian cells (including melanophorecells), or worm cells including cells of Caenorhabditis (includingCaenorhabditis elegans). Suitable mammalian cells include, for example,without limitation, COS cells (including Cos-1 and Cos-7), CHO cells,HEK293 cells, HEK293T cells, HEK293 T-Rex™ cells, or other transfectableeucaryotic cell lines. Suitable bacterial cells include withoutlimitation E. coli.

Preferably prokaryotes, such as E. coli, Bacillus, Streptomyces, ormammalian cells, like HeLa cells or Jurkat cells, or plant cells, likeArabidopsis, may be used.

Preferably the cell is an Aspergillus sp. or a fungal cell, preferably,it can be selected from the group consisting of the genera.Saccharomyces, Candida, Kluyveromyces, Hansenula, Schizosaccharomyces,Yarrowia, Pichia and Aspergillus.

Preferably the E. coli host cell is an E. coli host cell which isrecognized by the industry and regulatory authorities (including but notlimited to an E. coli K12 host cell or as demonstrated in the Examples,an E. coli BL21 host cell).

One preferred host cell to use with the present disclosure is E. coli,which may be recombinantly prepared as described herein. Thus, therecombinant host may be a recombinant E. coli host cell. There arelibraries of mutants, plasmids, detailed computer models of metabolismand other information available for E. coli, allowing for rationaldesign of various modules to enhance product yield. Methods similar tothose described above for Saccharomyces can be used to make recombinantE. coli microorganisms.

In one embodiment, the recombinant E. coli microorganism comprisesnucleotide sequences encoding SHC genes (as disclosed for example in anyone or more of Tables 10, 11 and 12 herein or functionalequivalents/homologies thereof including but not limited to variants,homologues mutants, derivatives or fragments thereof.

Preferably, the recombinant E. coli microorganism comprises a vectorconstruct as provided in FIGS. 5 and 21.

In another preferred embodiment, the recombinant K coil microorganismcomprises nucleotide sequences encoding WT SHC/HAC and WT SHC/HACderivatives genes or functional equivalents/homologies thereof includingbut not limited to variants, homologues mutants, derivatives orfragments thereof as set out in any one or more of Table 13, Table 14,Table 15, Table 16, Table 17 and/or Table 4a.

Another preferred host cell to use with the present disclosure is S.cerevisiae which is a widely used chassis organism in synthetic biology.Thus, the recombinant host may be S. cerevisiae. There are libraries ofmutants, plasmids, detailed computer models of metabolism and otherinformation available for S. cerevisiae, allowing for rational design ofvarious modules to enhance product yield. Methods are known for makingrecombinant S. cerevisiae microorganisms.

Culturing of cells is performed in a conventional manner. The culturemedium contains a carbon source, at least one nitrogen source andinorganic salts, and vitamins are added to it. The constituents of thismedium can be the ones which are conventionally used for culturing thespecies of microorganism in question.

Carbon sources of use in the instant method include any molecule thatcan be metabolized by the recombinant host cell to facilitate growthand/or production of (−)-Ambrox. Examples of suitable carbon sourcesinclude, but are not limited to, sucrose (eg., as found in molasses),fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose orother glucose containing polymer.

In embodiments employing yeast as a host, for example, carbon sourcessuch as sucrose, fructose, xylose, ethanol, glycerol, and glucose aresuitable. The carbon source can be provided to the host organismthroughout the cultivation period or alternatively, the organism can begrown for a period of time in the presence of another energy source,eg., protein, and then provided with a source of carbon only during thefed-batch phase.

The suitability of a recombinant host cell microorganism for use in themethods of the present disclosure may be determined by simple testprocedures using well known methods. For example, the microorganism tobe tested may be propagated in a rich medium (eg., LB-medium,Bacto-tryptone yeast extract medium, nutrient medium and the like) at apH, temperature and under aeration conditions commonly used forpropagation of the microorganism. Once recombinant microorganisms (i.e.recombinant host cells) are selected that produce the desired productsof bioconversion, the products are typically produced by a productionhost cell line on the large scale by suitable expression systems andfermentations, eg. by microbial production in cell culture.

In one embodiment of the present disclosure, a defined minimal mediumsuch as M9A is used for cell cultivation.

The components of M9A medium comprise: 14 g/l KH₂PO₄, 16 g/l K₂HPO₄, 1g/l Na₃Citrate.2H₂O, 7.5 g/l (NH₄)₂SO₄, 0.25 g/l MgSO₄.7H₂O, 0.015 g/lCaCl₂.2H₂O, 5 g/l glucose and 1.25 g/l yeast extract).

In another embodiment of the present disclosure, nutrient rich mediumsuch as LB was used. The components of LB medium comprise: 10 g/ltryptone, 5 g/l yeast extract, 5 g/l NaCl.

Other examples of Mineral Medium and M9 Mineral Medium are disclosed,for example, in U.S. Pat. No. 6,524,831B2 and US 2003/0092143A1.

The recombinant microorganism may be grown in a batch, fed batch orcontinuous process or combinations thereof. Typically, the recombinantmicroorganism is grown in a fermentor at a defined temperature(s) in thepresence of a suitable nutrient source, eg., a carbon source, for adesired period of time to produce sufficient enzyme to bioconverthomofarnesol to Ambrox and to produce a desired amount of Ambroxincluding (−)-Ambrox.

The recombinant host cells may be cultivated in any suitable manner, forexample by batch cultivation or fed-batch cultivation.

As used herein, the term “batch cultivation” is a cultivation method inwhich culture medium is neither added nor withdrawn during thecultivation.

As used herein, the term “fed-batch” means a cultivation method in whichculture medium is added during the cultivation but no culture medium iswithdrawn.

One embodiment of the present disclosure provides a method of producingAmbrox in a cellular system comprising expressing WT SHC or SHC/HACderivatives under suitable conditions in a cellular system, feedinghomofarnesol to the cellular system, converting homofarnesol to Ambroxusing the SHC or SHC/HAC derivatives produced using the cellular system,collecting Ambrox from cellular system and optionally isolating the(−)-Ambrox materials from the system. Expression of other nucleotidesequences may serve to enhance the method. The bioconversion method caninclude the additional expression of other nucleotide sequences in thecellular system. The expression of other nucleotide sequences mayenhance the bioconversion pathway for making (−)-Ambrox.

A further embodiment of the present disclosure is a bioconversion methodof making (−)-Ambrox comprising growing host cells comprising WT SHC/HACor SHC/HAC derivative genes, producing WT SHC/HAC or SHC/HAC derivativesin the host cells, feeding homofarnesol (eg. EEH) to the host cells,incubating the host cells under conditions of pH, temperature andsolubilizing agent suitable to promote the conversion of homofarnesol toAmbrox and collecting (−)-Ambrox. The production of the WT SHC/HACand/or SHC/HAC derivatives in the host cells provides a method of making(−)-Ambrox when homofarnesol is added to the host cells under suitablereaction conditions. Achieved conversion may be enhanced by adding morebiocatalyst and SDS to the reaction mixture.

The recombinant host cell microorganism may be cultured in a number ofways in order to provide cells in suitable amounts expressing the WT SHCor SHC/HAC derivatives for the subsequent bioconversion step. Since themicroorganisms applicable for the bioconversion step vary broadly (eg.yeasts, bacteria and fungi), culturing conditions are, of course,adjusted to the specific requirements of each species and theseconditions are well known and documented. Any of the art known methodsfor growing cells of recombinant host cell microorganisms may be used toproduce the cells utilizable in the subsequent bioconversion step of thepresent disclosure. Typically the cells are grown to a particulardensity (measurable as optical density (OD)) to produce a sufficientbiomass for the bioconversion reaction.

The cultivation conditions chosen influence not only the amount of cellsobtained (the biomass) but the quality of the cultivation conditionsalso influences how the biomass becomes a biocatalyst. The recombinanthost cell microorganism expressing the WT SHC or SHC/HAC derivative geneand producing the WT SHC or SHC/HAC derivative enzymes is termed abiocatalyst which is suitable for use in a bioconversion reaction. Insome embodiments the biocatalyst is a recombinant whole cell producingWT SHC or a SHC/HAC derivatives or it may be in suspension or animmobilized format. In other embodiments, the biocatalyst is a membranefraction or a liquid fraction prepared from the recombinant whole cellproducing the WT SHC or the SHC/HAC derivative (as disclosed for examplein Seitz et al 2012—as cited above).

The recombinant whole cell producing WT SHC or a SHC/HAC derivativesinclude whole cells collected from the fermenter (for the bioconversionreaction) or the cells in the fermenter (which are then used in aone-pot reaction). The recombinant whole cell producing WT SHC orSHC/HAC derivatives can include intact recombinant whole cell and/orcell debris. Either way, the WT SHC or SHC/HAC derivative is associatedwith a membrane (such as a cell membrane) in some way in order toreceive and/or interact with a substrate (eg. homofarnesol), whichmembrane (such as a cell membrane) can be part of or comprise a wholecell (eg. a recombinant whole cell). The WT SHC or SHC/HAC derivativesmay also be in an immobilized form (eg. associated with an enzymecarrier) which allows the WT SHC or SHC/HAC derivatives to interact witha substrate (eg. homofarnesol). The WT SHC or SHC/HAC derivatives mayalso be used in a soluble form.

In one embodiment, the biocatalyst is produced in sufficient amounts (tocreate a sufficient biomass), harvested and washed (and optionallystored (eg. frozen or lyophilized)) before the bioconversion step.

In a further embodiment, the cells are produced in sufficient amounts(to create a sufficient biocatalyst) and the reaction conditions arethen adjusted without the need to harvest and wash the biocatalyst forthe bioconversion reaction. This one step (or “one pot”) method isadvantageous as it simplifies the process while reducing costs. Theculture medium used to grow the cells is also suitable for use in thebioconversion reaction provided that the reaction conditions areadjusted to facilitate the bioconversion reaction.

The optimum pH for growing the cells is in the range of 6.0-7.0. Theoptimum pH for the bioconversion reaction is dependent on the type ofSHC/HAC enzyme used in the bioconversion reaction. The pH is regulatedusing techniques which are well known to the Skilled Person.

As Example 9 demonstrates, a “one pot” method was used to bioconverthomofarnesol to (−)-Ambrox with a 100% conversion rate. As Example 18demonstrates, a “one pot” method was used to bioconvert homofarnesol to(−)-Ambrox with a 99% conversion rate.

As used herein, any reference herein to a 99%/100% conversion rate for ahomofarnesol substrate to (−)-Ambrox is a reference to a 99%/100%conversion of the homofarnesol isomer (i.e. EEH) capable of conversionto (−)-Ambrox using a WT SHC/HAC or a SHC/HAC derivative enzyme.

Whilst the terms “mixture” or “reaction mixture” may be usedinterchangeably with the term “medium” in the present disclosure(especially as it relates to a “one pot” reaction), it should be notedthat growing the cells to create a sufficient biomass requires a cellculture/fermentation medium but a medium is not required for thebioconversion step as a reaction buffer will suffice at a suitable pH.

The bioconversion methods of the present disclosure are carried outunder conditions of time, temperature, pH and solubilizing agent toprovide for conversion of the homofarnesol feedstock to (−)-Ambrox. ThepH of the reaction mixture may be in the range of 4-8, preferably, 5 to6.5, more preferably 4.8-6.0 for the SHC/HAC derivative enzymes and inthe range of from about pH 5.0 to about pH 7.0 for the WT SHC enzyme andcan be maintained by the addition of buffers to the reaction mixture. Anexemplary buffer for this purpose is a citric acid buffer. The preferredtemperature is between from about 15° C. and about 45° C., preferablyabout 20° C. and about 40° C. although it can be higher, up to 55° C.for thermophilic organisms especially if the WT enzyme (eg. WT SHC/HAC)from the thermophilic microorganism is used. The temperature can be keptconstant or can be altered during the bioconversion process.

The Applicant has demonstrated that it may be useful to include asolubilizing agent (eg. a surfactant, detergent, solubility enhancer,water miscible organic solvent and the like) in the bioconversionreaction. As used herein, the term “surfactant” means a component thatlowers the surface tension (or interfacial tension) between two liquidsor between a liquid and a solid. Surfactants may act as detergents,wetting agents, emulsifiers, foaming agents, and dispersants. Examplesof surfactants include but are not limited to Triton X-100, Tween 80,taurodeoxycholate, Sodium taurodeoxycholate, Sodium dodecyl sulfate(SDS), and/or sodium lauryl sulfate (SLS).

Whilst Triton X-100 may be used to partially purify the WT SHC/HAC orSHC/HAC derivative enzyme (in soluble or membrane fraction/suspensionform), it may also be used in the bioconversion reaction (see forexample the disclosure in Seitz (2012 PhD thesis as cited above) as wellas the disclosure in Neumann and Simon (1986—as cited above) andJP2009060799.

However, surprisingly, as Example 14 demonstrates, the Applicantselected and identified SDS as a particularly useful solubilizing agentfrom a long list of other less useful solubilizing agents. Inparticular, the Applicant identified SDS as a remarkably bettersolubilizing agent than eg. Triton X-100 in terms of reaction velocityand yield for the homofarnesol to (−)-Ambrox bioconversion reaction(when EEH is used at both 4 g/l and 125 g/l). As demonstrated by thecomparative data in Example 12, the Applicant has demonstrated that forat least one SHC/HAC derivative enzyme, that maximal homofarnesol to(−)-Ambrox bioconversion activity with Triton X-100 (at a concentrationrange of about 0.005% to 0.48%) in the reaction was only around 20% ofthe activity obtained with SDS (at a concentration of about 0.07%).

Without wishing to be bound by theory, the use of SDS with recombinantmicrobial host cells may be advantageous as the SDS may interactadvantageously with the host cell membrane in order to make the SHCenzyme (which is a membrane bound enzyme) more accessible to thehomofarnesol substrate. In addition, the inclusion of SDS at a suitablelevel in the reaction mixture may improve the properties of the emulsion(homofarnesol in water) and/or improve the access of the homofarnesolsubstrate to the SHC enzyme within the host cell while at the same timepreventing the disruption (eg. denaturation of the SHC (WT or SHC/HACderivative) enzyme).

The concentration of the solubilising agent (eg. SDS) used in thebioconversion reaction is influenced by the biomass amount and thesubstrate (EEH) concentration. That is, there is a degree ofinterdependency between the solubilising agent (eg. SDS) concentration,the biomass amount and the substrate (EEH) concentration. By way ofexample, as the concentration of homofarnesol substrate increases,sufficient amounts of biocatalyst and solubilising agent (eg. SDS) arerequired for an efficient bioconversion reaction to take place. If, forexample, the solubilising agent (eg. SDS) concentration is too low, asuboptimal homofarnesol conversion may be observed. On the other hand,if, for example, the solubilising agent (eg. SDS) concentration is toohigh, then there may be a risk that the biocatalyst is affected througheither the disruption of the intact microbial cell and/ordenaturation/inactivation of the SHC/HAC enzyme.

The selection of a suitable concentration of SDS in the context of thebiomass amount and, substrate (EEH) concentration is within theknowledge of the Skilled Person. By way of example, a predictive modelis available to the Skilled Person to determine the suitable SDS,substrate (EEH) and biomass concentrations. By way of further example,Example 3 demonstrates that SDS in the range of 0.010-0.075% isappropriate when 4 g/l EEH and biocatalyst to an OD of 10.0 (650 nm) areused. Example 7 demonstrates that when 125 g/l EEH is used with 2× thewet weight of biomass, an adjusted SDS concentration (1.55%) isappropriate. However, an investigation of the percent EEH conversion to(−)-Ambrox using different SDS/cells ratio values indicated that thecorrect selection of the ratio of biocatalyst, homofarnesol substrateand solubilising agent (eg. SDS) facilitates the development of a robustbioconversion reaction system which demonstrates a degree of toleranceto a range of SDS concentrations (see for example FIG. 17) and pH ranges(see Example 15, FIG. 18).

The temperature of the bioconversion reaction for a WT SHC enzyme (eg,AacSHC) is from about 45-60° C., preferably 55° C.

The pH range of the bioconversion reaction for a WT SHC enzyme (eg.AacSHC) is from about 5.0 to 7.0, more preferably from about 5.6 toabout 6.2, even more preferably about 6.0.

The temperature of the bioconversion reaction for a SHC/HAC derivativeenzyme is about 34° C. to about 50° C., preferably about 35° C.

The pH of the bioconversion reaction for a SHC/HAC derivative enzyme isabout 4.8-6.4, preferably about 5.2-6.0.

Preferably the solubilising agent used in the bioconversion reaction isSDS.

The SDS concentration used in the bioconversion reaction for the WT SHCenzyme (eg. AacSHC) is in the range of about 0.010-0.075%, preferablyabout 0.030% when EEH at about 4 g/l is used.

The SDS concentration used in the bioconversion reaction for the SHC/HACderivative enzyme is in the range of about 0.0025-0.090%, preferablyabout 0.050% when EEH at about 4 g/l is used.

The biocatalyst is loaded to the reaction to an OD of 10.0 (650 nm) whenthe reaction is loaded with homofarnesol at an EEH concentration ofabout 4 g/l EEH.

The [SDS]/[cells] ratio is in the range of about, 10:1-20:1, preferablyabout 15:1-18:1, preferably about 16:1 when the ratio of biocatalyst toEEH homofarnesol is about 2:1 The SDS concentration in the bioconversionreaction for a SHC variant enzyme is in the range of about 1-2%,preferably in the range of about 1.4-1.7%, even more preferably about1.5% when the homofarnesol concentration is about 125 g/l EEH and thebiocatalyst concentration is 250 g/l (corresponding to an OD of about175 (650 nm)).

The ratio of biocatalyst to EEH homofarnesol substrate is in the rangeof about 0.5:1-2:1, in some embodiments 2:1, preferably about 1:1 or0.5:1.

In some embodiments, Ambrox is produced using a biocatalyst to which thehomofarnesol substrate is added. It is possible to add the substrate byfeeding using known means (eg. peristaltic pump, infusion syringe andthe like). Homofarnesol is an oil soluble compound and is provided in anoil format. Given that the biocatalyst (microbial cells such as intactrecombinant whole cell and/or cell debris and/or immobilised enzyme) ispresent in an aqueous phase, the bioconversion reaction may be regardedas a three phase system (comprising an aqueous phase, a solid phase andan oil phase) when homofarnesol is added to the bioconversion reactionmixture. This is the case even when SDS is present. By way ofclarification, when a soluble WT SHC or a SHC/HAC derivative is used asa biocatalyst, this is considered a two phase system.

The number of homofarnesol isomers present may influence the speed ofthe reaction. As Example 11 demonstrates, an SHC/HAC derivative enzymeis capable of biocoverting E,E-homofarnesol to (−)-Ambrox from a complexmixture of homofarnesol isomers (eg. EE:EZ:ZE:ZZ). However, a lowerconversion rate is typically observed which is consistent with the viewthat homofarnesol isomers other than EEH may compete with EEH for accessto the SHC/HAC derivative enzymes and thus may act as competitiveinhibitors for the conversion of EEH to (−)-Ambrox and/or also act asalternative substrates.

Accordingly, preferably the homofarnesol substrate comprises astereoisomeric mixture of 2-4 isomers, preferably two isomers.

Accordingly, preferably the homofarnesol substrate consists of orconsists essentially of a stereoisomeric mixture of 2-4 isomers,preferably two isomers.

Preferably the homofarnesol substrate comprises an EE:EZ stereoisomericmixture.

Preferably the homofarnesol substrate consists of or consistsessentially of an EE:EZ stereoisomeric mixture.

As Example 9 demonstrates, a 100% conversion of EE:EZ in a weight ratioof 87:13 was observed in a “one pot” fermentation and bioconversionreaction carried out over a period of 22.5 days. About 10 g of EEH wasconverted over this period of time.

As described in detail in Example 7, in a preferred embodiment, afermenter is used to grow recombinant host cell expressing the SHC/HACderivative gene and producing active SHC/HAC derivative enzymes to asufficient biomass concentration suitable for use as a biocatalyst inthe same fermenter vessel which is used to convert the homofarnesolsource to (−)-Ambrox in admixture with one or more of the by-products(II), (IV) and/or (III as disclosed, for example, in FIG. 12). Theresulting (−)-Ambrox may be isolated by steam extraction/distillation ororganic solvent extraction using a non-water miscible solvent (toseparate the reaction products and unreacted substrate from thebiocatalyst which stays in the aqueous phase) followed by subsequentevaporation of the solvent to obtain a crude reaction product asdetermined by gas chromatographic (GC) analysis. The steamextraction/distillation and organic solvent extraction methods are knownto those skilled in the art.

By way of example, the resulting (−)-Ambrox may be extracted from thewhole reaction mixture using an organic solvent such as a non-watermiscible solvent (for example toluene). Alternatively, the resulting(−)-Ambrox may be extracted from the solid phase of the reaction mixture(obtained by, for example, centrifugation or filtration) using a watermiscible solvent (for example ethanol) or a non-water miscible solvent(for example toluene). By way of further example, (−)-Ambrox is presentin the solid phase as crystals or in amorphous form and can be separatedfrom the remaining solid phase (cell material or debris thereof) and theliquid phase also by means of filtration. By way of further example, ata temperature above the melting point of (−)-Ambrox (around, 75° C.),the (−)-Ambrox may form an oil layer on top of aqueous phase, which oillayer can be removed and collected. In order to ensure a completerecovery of (−)-Ambrox after the oil layer is removed, an organicsolvent may be added to the aqueous phase containing the biomass inorder to extract any residual (−)-Ambrox contained in, or on or aboutthe biomass. The organic layer can be combined with the oil layer,before the whole is further processed to isolate and purify (−)-Ambrox.

The (−)-Ambrox may be further selectively crystallised to removeby-products (II), (IV) and (III) and any unreacted homofarnesolsubstrate from the final (−)-Ambrox product. The term “selectivecrystallization” refers to a process step whereby (−)-Ambrox is causedto crystallise from a solvent whilst the compounds (II), (III) and (IV)remain dissolved in the crystallizing solvent to such an extent thatisolated crystalline material contains only (−)-Ambrox product, or if itcontains any of the other compounds (II), (III) or (IV), then they arepresent only in olfactory acceptable amounts.

The selective crystallisation step may use a water miscible solvent suchas ethanol or the like. The olfactive purity of the final (−)-Ambroxproduct is determined using a 10% ethanol extract in water or by testingthe crystalline material. The final (−)-Ambrox product is tested againsta commercially available reference of (−)-Ambrox product for itsolfactive purity, quality and its sensory profile. The (−)-Ambroxmaterial is also tested in application studies by experts in order todetermine if the material meets the specifications with respect to itsorganoleptic profile. Various applications for (−)-Ambrox include butare not limited to a fine fragrance or a consumer product such as fabriccare, toiletries, beauty care and cleaning products includingessentially all products where the currently available Ambroxingredients are used commercially, including but not limited to: Ambrox(Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest),Cetalox Laevo (Firmenich), Ambermor (Aromor) and Norambrenolide Ether(Pacific) products.

The selective crystallisation of (−)-Ambrox may be influenced by thepresence of unreacted homofarnesol substrate and also the ratio of(−)-Ambrox to the other detectable by-products (II), (III) and/or (IV).Even if only 10% conversion of the homofarnesol substrate to (−)-Ambroxis obtained (as demonstrated in Example 7 using a WT SHC/HAC enzyme),the selective crystallisation of (−)-Ambrox is still possible.

Examples of suitable water miscible and non-water miscible organicsolvents suitable for use in the extraction and/or selectivecrystallization of (−)-Ambrox include but are not limited to aliphatichydrocarbons, preferably those having 5 to 8 carbon atoms, such aspentane, cyclopentane, hexane, cyclohexane, heptane, octane orcyclooctane, halogenated aliphatic hydrocarbons, preferably those havingone or two carbon atoms, such as dichloromethane, chloroform, carbontetrachloride, dichloroethane or tetrachloroethane, aromatichydrocarbons, such as benzene, toluene, the xylenes, chlorobenzene ordichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols,preferably those having 4 to 8 carbon atoms, such as ethanol,isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butylether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuranor esters such as ethyl acetate or n-butyl acetate or ketones such asmethyl isobutyl ketone or dioxane or mixtures of these. The solventswhich are especially preferably used are the abovementioned heptane,Methyl tert-butyl ether (also known as MTBE, tert-butyl methyl ether,tertiary butyl methyl ether and tBME), diisopropyl ether,tetrahydrofuran, ethyl acetate and/or mixtures thereof.

Preferably, a water miscible solvent such as ethanol is used for theextraction of (−)-Ambrox from the solid phase of the reaction mixture.The use of ethanol is advantageous because it is easy to handle, it isnon toxic and it is environmentally friendly.

The term “isolated” as used herein refers to a bioconversion productsuch as (−)-Ambrox which has been separated or purified from componentswhich accompany it. An entity that is produced in a cellular systemdifferent from the source from which it naturally originates is“isolated”, because it will necessarily be free of components whichnaturally accompany it. The degree of isolation or purity can bemeasured by any appropriate method, eg., gas chromatography (GC), HPLCor NMR analysis.

In some embodiments, the end product ((−)-Ambrox) is isolated andpurified to homogeneity (eg., at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, or 89.5% pure or 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 99.5% pure).

Desirably, the amount of (−)-Ambrox produced can be from about 1 mg/l toabout 20,000 mg/l (20 g/l) or higher such as from about 20 g/l to about200 g/l or from 100-200 g/l, preferably about 125 g/l or 150 g/l orabout 188 g/l.

As Example 7 demonstrates, at least 125 g/l (−)-Ambrox is produced in abioconversion reaction using a recombinant E. coli host cell producing aSHC/HAC derivative enzyme over about 2 days.

As Example 19 demonstrates, it is possible to run bioconversions at 188g/l EEH or higher provided efficient mixing is achieved as stirringefficiency appears to be the only limitation of the system. In addition,a biocatalyst with improved activity (eg. in terms of SHC variants withfurther improved activity or in terms of increased SHC enzymeproduction) may improve or maintain productivity using less biomasswhich is advantageous with respect to mixing efficiencies.

For example about 1 to about 100 mg/l, about 30 to about 100 mg/l, about50 to about 200 mg/l, about 100 to about 500 mg/l, about 100 to about1,000 mg/l, about 250 to about 5,000 mg/l, about 1,000 (1 g/l) to about15,000 mg/l (15 g/l), or about 2,000 (2 g/l) to about 10,000 mg/l (10g/l) or about 2,000 (2 g/l) to about 25,000 mg/l (25 g/l) or about 2,000(2 g/l) to about 25,000 mg/l (25 g/l), 26,000 mg/l (26 g/l), 27,000 mg/l(27 g/l), 28,000 mg/l (28 g/l), 29,000 mg/l (29 g/l), 30,000 mg/l (30g/l), 40 g/l, 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, 100 g/l, 110 g/l,120 g/l, 125 g/l, 130 g/l, 140 g/l, 150 g/l, 160 g/l, 170 g/l, 180 g/l,190 g/l or 200 g/l or 300 g/l or 400 g/l or 500 g/l of (−)-Ambrox isproduced.

Preferably (−)-Ambrox at a concentration of at least 100 g/l is producedwithin a period of time of from 48 to 72 hours.

Preferably (−)-Ambrox at a concentration of about 150 g/l is producedwithin a time period of from about 48 to 72 hours.

Preferably (−)-Ambrox at a concentration of about 200 g/l is producedwithin a time period of from about 48 to 72 hours.

Preferably (−)-Ambrox at a concentration of about 250 g/l is producedwithin a time period of from about 48 to 72 hours.

The Skilled Person will understand that higher cumulative productiontiters can be achieved by implementing a continuous process, such asproduct removal, substrate feed, and biomass addition or (partial)replacement.

Preferably the bioconversion of EEH into (−)-Ambrox in the presence of arecombinant host cell comprising a WT SHC/HAC or a SHC/HAC derivativegenerates an Ambrox yield of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, given in mol percentand based on the mols of EEH employed; especially preferably, the yieldis between 5 and 100, 10 and 100, and 100, 25 and 100, 30 and 100, 35and 100, in particular between 40 and 100, 45 and 100, 50 and 100, 60and 100, 70 and 100 mol percent.

The activity of the SHC/HAC enzyme is defined via the reaction rate(amount of product/(amount of product+amount of remaining startingmaterial))×100) in mol percent. Preferably, the bioconversion of EEHinto (−)-Ambrox in the presence of WT SHC or a SHC/HAC derivative enzymeproduces an (−)-Ambrox yield of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, given in molpercent and based on the mols of EEH employed; especially preferably,the yield is between 5 and 100, 10 and 100, 20 and 100, 25 and 100, 30and 100, 35 and 100, in particular between 40 and 100, 45 and 100, 50and 100, 60 and 100, 70 and 100.

In a preferred embodiment of the invention, the yield and/or thereaction rate are determined over a defined time period of, for example,4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, during which EEH isconverted into (−)-Ambrox by a recombinant host cell comprising anucleotide sequence encoding a WTSHC or the SHC/HAC derivative enzymeaccording to the present disclosure. In a further variant, the reactionis carried out under precisely defined conditions of, for example, 25°C., 30° C., 40° C., 50° C. or 60° C. In particular, the yield and/or thereaction rate are determined by carrying out the reaction of convertingEEH into (−)-Ambrox by the SHC/HAC derivative enzymes according to theinvention at 35° C. over a period of 24-72 hours.

In a further embodiment of the present invention, a recombinant hostcell comprising a nucleotide sequence encoding a SHC/HAC derivative ischaracterized in that it shows a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-,11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-,25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-,39-, 40-, 41-, 42-, 43-, 44-, 45-, 46-, 47-, 48-, 49-, 50-, 51-, 52-,53-, 54-, 55-, 56-, 57-, 58-, 59-, 60-, 61-, 62-, 63-, 64-, 65-, 66-,67-, 68-, 69-, 70-, 71-, 72-, 73-, 74-, 75-, 76-, 77-, 78-, 79-, 80-,81-, 82-, 83-, 84-, 85-, 86-, 87-, 88-, 89-, 90-, 91-, 92-, 93-, 94-,95-, 96-, 97-, 98-, 99-, 100-, 200-, 500-, 1000-fold or higher yieldand/or reaction rates in the reaction of homofarnesol to give (−)-Ambroxin comparison with the WT SHC or SHC/HAC derivative enzyme under thesame conditions. Here, the term condition relates to reaction conditionssuch as substrate concentration, enzyme concentration, reaction periodand/or temperature.

The successful development of a bioconversion process for making(−)-Ambrox from homofarnesol in a recombinant strain of E. colicomprising a nucleotide sequence encoding a WT/reference SHC or aSHC/HAC derivative can offer a low cost and industrially economicalprocess for (−)-Ambrox production.

As Example 7 demonstrates, the present disclosure provides for a 100%conversion of E,E-Homofarnesol (125 g/l) to (−)-Ambrox after 48 hours ofincubation with an optimised SHC/HAC Derivative with a 8-foldimprovement in yield when an AacSHC derivative is used compared with theWT AacSHC enzyme (see FIG. 11).

Functional homologs of the WT Reference SHC/HAC or the SHC/HACderivative polypeptides described herein are also suitable for use inproducing Ambrox in a recombinant host. Thus, the recombinant host mayinclude one or more heterologous nucleic acid(s) encoding functionalhomologs of the polypeptides described above and/or a heterologousnucleic acid encoding a SHC/HAC derivative enzyme as described herein.

A functional homolog is a polypeptide that has sequence similarity to areference polypeptide, and that carries out one or more of thebiochemical or physiological function(s) of the reference polypeptide. Afunctional homolog and the reference polypeptide may be naturaloccurring polypeptides, and the sequence similarity may be due toconvergent or divergent evolutionary events. As such, functionalhomologs are sometimes designated in the literature as homologs, ororthologs, or paralogs. Variants of a naturally occurring functionalhomolog, such as polypeptides encoded by mutants of a wild-type codingsequence, may themselves be functional homologs. Functional homologs canalso be created via site-directed mutagenesis of the coding sequence fora polypeptide, or by combining domains from the coding sequences fordifferent naturally-occurring polypeptides (“domain swapping”).Techniques for modifying genes encoding functional homologs describedherein are known and include, inter alia, directed evolution techniques,site-directed mutagenesis techniques and random mutagenesis techniques,and can be useful to increase specific activity of a polypeptide, altersubstrate specificity, alter expression levels, alter subcellularlocation, or modify polypeptide:polypeptide interactions in a desiredmanner. Such modified polypeptides are considered functional homologs.The term “functional homolog” is sometimes applied to the nucleic acidthat encodes a functionally homologous polypeptide.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofthe nucleic acid sequences encoding the SHC derivative polypeptides andthe like.

Hybridization can also be used to identify functional homologs and/or asa measure of homology between two nucleic acid sequences. A nucleic acidsequence encoding any of the proteins disclosed herein, or a portionthereof, can be used as a hybridization probe according to standardhybridization techniques. The hybridization of a probe to DNA or RNAfrom a test source (eg., a mammalian cell) is an indication of thepresence of the relevant DNA or RNA in the test source. Hybridizationconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y.,63.1-6.3.6, 1991. Moderate hybridization conditions are defined asequivalent to hybridization in 2× sodium chloride/sodium citrate (SSC)at 30° C. followed by a wash in 1×SSC, 0.1% SDS at 50° C. Highlystringent conditions are defined as equivalent to hybridization in 6×sodium chloride/sodium citrate (SSC) at 45° C. followed by a wash in0.2×SSC, 0.1% SDS at 65° C.

Sequence analysis to identify functional homologs can also involveBLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundantdatabases using a relevant amino acid sequence as the referencesequence. Amino acid sequence is, in some instances, deduced from thenucleotide sequence. Those polypeptides in the database that havegreater than 40% sequence identity are candidates for further evaluationfor suitability for use in the SHC/HAC bioconversion reaction. Aminoacid sequence similarity allows for conservative amino acidsubstitutions, such as substitution of one hydrophobic residue foranother or substitution of one polar residue for another. If desired,manual inspection of such candidates can be carried out in order tonarrow the number of candidates to be further evaluated. Manualinspection can be performed by selecting those candidates that appear tohave for eg., conserved functional domains.

Typically, polypeptides that exhibit at least about 30% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related polypeptides exhibit at least 30%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, aminoacid sequence identity. In some embodiments, a conserved region exhibitsat least, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% amino acidsequence identity. Sequence identity can be determined as set forthabove and below.

The produced WTSHC and/or SHC/HAC Derivative is based on an amino acidSEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or avariant, homologue, mutant, derivative or fragment thereof.

The produced SHC is based on an amino acid sequence with at least 30%,40%, 41%, 42%, 43%, 44% 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4.

In addition, the produced reference SHC is based on an amino acidsequence produced from E. coli.

“Percent (%) identity” with respect to the nucleotide sequence of a geneis defined as the percentage of nucleotides in a candidate DNA sequencethat is identical with the nucleotides in the DNA sequence, afteraligning the sequence and introducing gaps, if necessary, to achieve themaximum percent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent nucleotide sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared.

The terms “polypeptide” and “protein” are used interchangeably hereinand mean any peptide-linked chain of amino acids, regardless of lengthor post-translational modification.

As used herein the term “derivative” includes but is not limited to avariant. The terms “derivative” and “variant” are used interchangeablyherein.

As used herein, the term “variant” is to be understood as a polypeptidewhich differs in comparison to the polypeptide from which it is derivedby one or more changes in the amino acid sequence. The polypeptide fromwhich a variant is derived is also known as the parent or referencepolypeptide. Typically a variant is constructed artificially, preferablyby gene-technological means. Typically, the polypeptide from which thevariant is derived is a wild-type protein or wild-type protein domain.However, the variants usable in the present disclosure may also bederived from homologs, orthologs, or paralogs of the parent polypeptideor from artificially constructed variants, provided that the variantexhibits at least one biological activity of the parent polypeptide. Thechanges in the amino acid sequence may be amino acid exchanges,insertions, deletions, N-terminal truncations, or C-terminaltruncations, or any combination of these changes, which may occur at oneor several sites.

In preferred embodiments, a variant usable in the present disclosureexhibits a total number of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) changes in theamino acid sequence (i.e. exchanges, insertions, deletions, N-terminaltruncations, and/or C-terminal truncations). The amino acid exchangesmay be conservative and/or non-conservative. In preferred embodiments, avariant usable in the present disclosure differs from the protein ordomain from which it is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 amino acid exchanges, preferably conservative amino acid changes.Variants may additionally or alternatively comprise deletions of aminoacids, which may be N-terminal truncations, C-terminal truncations orinternal deletions or any combination of these. Such variants comprisingN-terminal truncations, C-terminal truncations and/or internal deletionsare referred to as “deletion variants” or “fragments” in the context ofthe present application. The terms “deletion variant” and “fragment” areused interchangeably herein. A deletion variant may be naturallyoccurring (eg. splice variants) or it may be constructed artificially,preferably by gene-technological means. Typically, the protein orprotein domain from which the deletion variant is derived is a wild-typeprotein. However, the deletion variants of the present disclosure mayalso be derived from homologs, orthologs, or paralogs of the parentpolypeptide or from artificially constructed variants, provided that thedeletion variants exhibit at least one biological activity of the parentpolypeptide. Preferably, a deletion variant (or fragment) has a deletionof up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids at its N-terminusand/or at its C-terminus and/or internally as compared to the parentpolypeptide.

A “variant” as used herein, can alternatively or additionally becharacterised by a certain degree of sequence identity to the parentpolypeptide from which it is derived.

A variant of the WT/reference SHC/HAC or the SHC/HAC Derivative of thepresent disclosure may have a sequence identity of at least 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to the respectivereference polypeptide or to the respective reference polynucleotide.

The expression “at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%0, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity” is used throughout the specificationwith regard to polypeptide and polynucleotide sequence comparisons.

A polynucleotide belonging to a family of any of the enzymes disclosedherein or a protein can be identified based on its similarity to therelevant gene or protein, respectively. For example, the identificationcan be based on sequence identity. In certain preferred embodiments thedisclosure features isolated nucleic acid molecules which are at least30%, 40%, 41%, 42%, 43%, 44%,%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58% 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to (a)a nucleic acid molecule that encodes the polypeptide of SEQ ID No. 5-163(see Tables 14-17 and Table 4a as provided herein) (b) the nucleotidesequence of SEQ ID No. 6-168, 169, 170, 172, 174 and 176 (see Tables14-17 and Table 4a as provided herein) and (c) a nucleic acid moleculewhich includes a segment of at least 30 (eg., at least 30, 40, 50, 60,80, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 850,900, 950, 1000, or 1010) nucleotides of SEQ ID No. 6-168, 169, 170, 172,174 and 176 (see Tables 14-17 and Table 4a as provided herein).

Preferably, the polypeptide in question and the reference polypeptideexhibit the indicated sequence identity over a continuous stretch of 20,30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, thepolynucleotide in question and the reference polynucleotide exhibit theindicated sequence identity over a continuous stretch of 60, 90, 120,135, 150, 180, 210, 240, 270, 300 or more nucleotides. In case where twosequences are compared and the reference sequence is not specified incomparison to which the sequence identity percentage is to becalculated, the sequence identity is to be calculated with reference tothe longer of the two sequences to be compared, if not specificallyindicated otherwise. If the reference sequence is indicated, thesequence identity is determined on the basis of the full length of thereference sequence indicated by SEQ ID No. 1, 2, 3 and/or 4 if notspecifically indicated otherwise.

For example, a peptide sequence consisting of 130 amino acids comparedto the amino acids of full length of reference SHC with 631 amino acidresidues may exhibit a maximum sequence identity percentage of 20.6%(130/631×100) while a sequence with a length of 300 amino acids mayexhibit a maximum sequence identity percentage of 47.5% (300/631×100).The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, preferably with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package or with the CLUSTAL algorithm(Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic AcidsRes. 22, 4673-80) or the GAP program (mathematical algorithm of theUniversity of Iowa) as utilised in the sequence alignments to WTSHC asprovided in Table 18 herein or the mathematical algorithm of Myers andMiller (1989—Cabios 4: 11-17) as disclosed in and as utilised in theWTSHC sequence alignments in Table 19 as provided herein.

The grade of sequence identity (sequence matching) may be calculatedusing eg. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et a (1990)J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performedwith the BLASTN program, score=100, word length=12, to obtainpolynucleotide sequences that are homologous to those nucleic acidswhich encode the relevant protein.

BLAST protein searches are performed with the BLASTP program, score==50,word length===3, to obtain amino acid sequences homologous to the SrKOpolypeptide. To obtain gapped alignments for comparative purposes,Gapped BLAST is utilized as described in Altschul et al (1997) NucleicAcids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:154-162) or Markov random fields. When percentages ofsequence identity are referred to in the present application, thesepercentages are calculated in relation to the full length of the longersequence, if not specifically indicated otherwise.

Sensory

The bioconversion of homofarnesol to (−)-Ambrox according to the presentdisclosure produces (−)-Ambrox as a predominant compound but may alsoproduce compounds other than (−)-Ambrox which may or may not impartpleasant olfactive notes to the bioconversion mixture and so maycontribute in a positive or negative manner to the sensory character ofthe (−)-Ambrox end product. Accordingly a sensory analysis is carriedout using well established sensory tests utilized by trained Experts(eg. Perfumers) so that the testing can assist in determining if achemically relevant target product is also an olfactively relevant endproduct relative to a reference product. As the sensory analysis inExample 22 demonstrates, the removal of one of more by-product compoundsfrom (−)-Ambrox can improve the odor of the remaining compound((−)-Ambrox) even if the removed compounds are actually odorlesscompounds per se. That is, an (−)-Ambrox odor enhancement was observedin the absence of compounds II, III and IV.

Aspects of the Invention

1. A process for preparing (−)-Ambrox or a mixture comprising(−)-Ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture ofstereoisomers comprising EEH is enzymatically converted to (−)-Ambrox ora mixture comprising (−)-Ambrox wherein the enzymatic conversion iscarried out using an SHC/HAC enzyme under reaction conditions suitablefor the production of (−)-Ambrox and wherein the mixture ofstereoisomers comprising EEH consists essentially of homofarnesolisomers selected from the group consisting of [(3E,7E) and [(3Z,7E)]and/or [(3E,7E) and (3E,7Z)] and/or [(3Z,7E), (3E,7E) and (3E,7Z)] alsodesignated as [EE:EZ], [EE:ZE] and [EE:EZ:ZE] respectively.

2. A process for preparing (−)-Ambrox or a mixture comprising(−)-Ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture ofstereoisomers comprising EEH is converted enzymatically to give(−)-Ambrox or a mixture comprising (−)-Ambrox wherein the enzymaticconversion using an SHC/HAC enzyme is carried out under reactionconditions suitable for the production of (−)-Ambrox and wherein if thereaction is carried out in the presence of a solubilizing agent, TritonX-100 or Taurodeoxycholate is not used in combination with a wild-typeSHC/HAC enzyme.

3. A process for preparing (−)-Ambrox or a mixture comprising(−)-Ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture ofstereoisomers comprising EEH is enzymatically converted to (−)-Ambrox ora mixture comprising (−)-Ambrox wherein the enzymatic conversion iscarried out using an SHC/HAC enzyme under reaction conditions suitablefor the production of (−)-Ambrox and wherein the mixture ofstereoisomers comprising EEH consists essentially of homofarnesolisomers selected from the group consisting of [(3E,7E) and [(3Z,7E)]and/or [(3E,7E) and (3E,7Z)] and/or [(3Z,7E), (3E,7E) and (3E,7Z)] alsodesignated as [EE:EZ], [EE:ZE] and [EE:EZ:ZE] respectively and whereinthe reaction takes place in a three-phase system comprising an aqueousphase, a solid phase and an oil phase.

4. The process according to paragraph 1 or paragraph 2 or paragraph 3wherein the process is carried out using an SHC/HAC enzyme polypeptidesequence selected from the group consisting of SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3; SEQ ID No. 4, or a SHC/HAC derivative selected fromTable 1, Table 5, Table 2, Table 6, Table 3, Table 7. Table 4, Table 8or Table 13, Table 14, or selected from SEQ ID No. 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 171, 173, 175, 177and/or 178 or a sequence with at least 30% identity, at least 40%identity, at least 50% identity, or at least 60% identity, or at least70% identity, or at least 80% identity, or at least 90% identity, or atleast 95% identity, or at least 96% identity, or at least 97% identity,or at least 98% identity, or at least 99% identity to SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3, or SEQ ID No. 4.

5. The process of any one of paragraphs 1-4 wherein the process usesrecombinant host cells producing the SHC/HAC enzyme.

6. The process according to paragraph 4 or paragraph 5 wherein thenucleotide sequence encoding the SHC/HAC enzyme is selected from thegroup consisting of SEQ ID No. 165, 166, 167, 168, 169 or SEQ ID No. 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and/or170, 172, 174 and/or 176.

7. The process according to any one of paragraphs 1-6 wherein theconversion of homofarnesol to (−)-Ambrox takes place at a temperature inthe range of from 30° C. to 60° C., at a pH in the range of about 4-8.

8. The process according to any one of paragraphs 1-7 wherein theconversion of homofarnesol to (−)-Ambrox takes place using one or moreof the reaction conditions for the wild-type SHC/HAC or SHC/HACderivative enzymes as set out in Table 24 or Table 24a, preferably at apH range of 5.0 to 6.2, preferably at a temperature of 35° C.

9. The process according to any one of paragraphs 3-8 wherein theSDS/cell ratio in the range of 10:1 to 20:1, preferably at 16:1 when theratio of biocatalyst to EEH is about 2:1.

10. The process according to any one of paragraphs 3-9 wherein theweight ratio of biocatalyst to homofarnesol is in the range of fromabout 0.5-2:1, preferably about 1:1 or 05:1.

11. The process according to any one of paragraphs 3-10 wherein the cellgrowth and bioconversion reaction steps are carried out in the samereaction vessel.

12. The process according to paragraph 2 wherein the homofarnesolsubstrate comprises one or more homofarnesol stereoisomers.

13. The process according to paragraph 12 wherein the homofarnesolsubstrate comprises or consists essentially of two homofarnesolstereoisomers.

14. The process according to paragraph 13 wherein the homofarnesolsubstrate comprises or consists essentially of EE:EZ stereoisomers.

15. The process according to any one of paragraph 14 wherein thehomofarnesol comprises or consists essentially of an EE:EZ stereoisomermixture in the weight ratios selected from the group consisting of;100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09;90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19;80:20; 79:21; 78:22; 77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:2970:30; 69:31; 68:32; 67:33; 66:34; 65:35; 64:36; 63:37; 62:38; 61:39;and 60:40.

16. The process according to paragraph 15 wherein the homofarnesolcomprises or consists essentially of an EE:EZ stereoisomer mixture in aweight ratio selected from the group consisting of: EE:EZ 92:8; EE:EZ90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and EE:EZ66:34.

17. The process according to paragraph 15 or paragraph 16 wherein thehomofarnesol comprises or consists essentially of an EE:EZ stereoisomermixture in a weight ratio of 80:20.

18. The process according to any one of paragraphs 1-17 wherein(−)-Ambrox is produced in admixture with at least one or more of theby-products (II), (IV) and/or (III).

19. The process according to any one of paragraphs 1-18 wherein(−)-Ambrox is isolated from the bioconversion reaction mixture using anorganic solvent or a steam extraction/distillation step or filtration.

20. The process according to any one of paragraphs 1-19 wherein(−)-Ambrox is isolated from the solid phase of the bioconversionreaction mixture using an organic solvent or a steamextraction/distillation step.

21. The process according to paragraph 19 or paragraph 20 wherein the(−)-Ambrox is isolated from the reaction mixture using an organicsolvent.

22. The process according to paragraph 21 wherein (−)-Ambrox is isolatedfrom the reaction mixture using ethanol or toluene.

23. The process according to any one of paragraphs 19-22 wherein the(−)-Ambrox is selectively crystallized using an organic solvent.

24. The process according to paragraph 23 wherein the (−)-Ambrox issubstantially free of the by-products (II), (IV) and/or (III).

25. The process according to any one of paragraphs 1-24 wherein(−)-Ambrox in a concentration range of about 125-200 g/l is produced.

26. (−)-Ambrox obtainable by the method of any one of paragraphs 1-25wherein the (−)-Ambrox has an odor threshold of from about 0.1 to about0.5 ng/l.

27. The (−)-Ambrox of paragraph 26 in a solid form, preferably anamorphous or crystalline form.

28. A process for making a product containing (−)-Ambrox comprisingincorporating the (−)-Ambrox of any one of paragraph 26 or paragraph 27into the product.

29. The process of paragraph 28 wherein the product is a fragranceproduct, a cosmetic, a cleaning product, a detergent product or a soapproduct.

30. A fragrance or cosmetic or a consumer care product comprising the(−)-Ambrox of any one of paragraph 26 or paragraph 27.

31. A fragrance or cosmetic or consumer care composition comprising the(−)-Ambrox of paragraph 26 or paragraph 27 and one or more additionalcomponents.

32. The use of the (−)-Ambrox of paragraph 26 or paragraph 27 as part ofa fragrance or a cosmetic or a consumer product such as a fabric care,toiletry, beauty care and/or a cleaning product.

33. A process for augmenting, enhancing or imparting an aroma in or to afragrance composition comprising the step of admixing with saidfragrance composition, an aroma augmenting or enhancing product producedaccording to a process comprising the steps of:

(a) preparing a reaction mixture comprising (−)-Ambrox in admixture withone or more of the by-product compounds (II), (III) or (IV).

(b) extracting (−)-Ambrox in admixture with one or more of theby-product compounds (II), (III) or (IV); and

(c) selectively crystallizing (−)-Ambrox from the extraction mixture;

wherein the (−)-Ambrox is prepared by an enzymatic conversion of(3E,7E)-homofarnesol (EEH) or a mixture of stereoisomers comprising EEHusing an SHC/HAC enzyme under reaction conditions suitable for theproduction of (−)-Ambrox and wherein the mixture of stereoisomerscomprising EEH consists essentially of homofarnesol isomers selectedfrom the group consisting of [(3E,7E) and [(3Z,7E)] and/or [(3E,7E) and(3E,7Z)] and/or [(3Z,7E), (3E,7E) and (3E,7Z)] also designated as[EE:EZ], [EE:ZE] and [EE:EZ:ZE] respectively.

34. The process of paragraph 33 wherein the reaction takes place in athree-phase system comprising an aqueous phase, a solid phase and an oilphase.

35. The process according to paragraph 33 or paragraph 34 wherein theprocess is carried out using an SHC/HAC enzyme polypeptide sequenceselected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3; SEQ ID No. 4, or a SHC/HAC derivative selected from Table 1,Table 5, Table 2, Table 6, Table 3, Table 7, Table 4, Table 8 or Table14, or selected from SEQ ID No. 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 171, 173, 175 and/or 177 or a sequence withat least 30% identity, at least 40% identity, at least 50% identity, orat least 60% identity, or at least 70% identity, or at least 80%identity, or at least 90% identity, or at least 95% identity, or atleast 96% identity, or at least 97% identity, or at least 98% identity,or at least 99% identity to SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, orSEQ ID No. 4.

36. The process of any one of paragraphs 33-35 wherein the process usesrecombinant host cells producing the SHC/HAC enzyme.

37. The process according to paragraph 35 or paragraph 36 wherein thenucleotide sequence encoding the SHC/HAC enzyme is selected from thegroup consisting of SEQ ID No. 165, 166, 167, 168, 169 or SEQ ID No. 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and/or170, 172, 174 and/or 176.

38. The process according to any one of paragraphs 33-37 wherein theconversion of homofarnesol to (−)-Ambrox takes place at a temperature inthe range of from 30° C. to 60° C., at a pH in the range of about 4-8.

39. The process according to any one of paragraphs 33-38 wherein theconversion of homofarnesol to (−)-Ambrox takes place using one or moreof the reaction conditions for the wild-type SHC/HAC or SHC/HACderivative enzymes as set out in Table 24 or Table 24a, preferably at apH range of 5.0 to 6.2, preferably at a temperature of 35° C.

40. The process according to any one of paragraphs 34-39 wherein theSDS/cell ratio in the range of 10:1 to 20:1, preferably at 16:1 when theratio of biocatalyst to EEH is about 2:1.

41. The process according to any one of paragraphs 34-40 wherein theweight ratio of biocatalyst to homofarnesol is in the range of fromabout 0.5-2:1, preferably about 1:1 or 05:1.

42. The process according to any one of paragraphs 34-41 wherein thecell growth and bioconversion reaction steps are carried out in the samereaction vessel.

43. The process according to any one of paragraphs 33-42 wherein thehomofarnesol comprises or consists essentially of an EE:EZ stereoisomermixture in the weight ratios selected from the group consisting of:100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09;90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19;80:20; 79:21; 78:22; 77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29;70:30; 69:31; 68:32; 67:33; 66:34; 65:35; 64:36; 63:37; 62:38; 61:39;and 60:40.

44. The process according to paragraph 43 wherein the homofarnesolcomprises or consists essentially of an EE:EZ stereoisomer mixture in aweight ratio selected from the group consisting of EE:EZ 92:08; EE:EZ90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and EE:EZ66:34.

45. The process according to paragraph 43 or paragraph 44 wherein thehomofarnesol comprises or consists essentially of an EE:EZ stereoisomermixture in a weight ratio of 80:20.

46. The process according to any one of paragraphs 33-45 wherein(−)-Ambrox is produced in admixture with at least one or more of theby-products (II), (IV) and/or (III).

47. The process according to any one of paragraphs 33-46 wherein(−)-Ambrox is isolated from the bioconversion reaction mixture using anorganic solvent or a steam extraction/distillation step or filtration.

48. The process according to any one of paragraphs 33-47 wherein(−)-Ambrox is isolated from the solid phase of the bioconversionreaction mixture using an organic solvent or a steamextraction/distillation step.

49. The process according to paragraph 47 or paragraph 48 wherein the(−)-Ambrox is isolated from the reaction mixture using an organicsolvent.

50. The process according to paragraph 49 wherein (−)-Ambrox is isolatedfrom the reaction mixture using ethanol or toluene.

51. The process according to any one of paragraphs 47-49 wherein the(−)-Ambrox is selectively crystallized using an organic solvent.

52. The process according to paragraph 51 wherein the (−)-Ambrox issubstantially free of the by-products (II), (IV) and/or (III).

53. The process according to any one of paragraphs 33-52 wherein(−)-Ambrox in a concentration range of about 125-200 g/l is produced.

54. The process according to any one of paragraphs 33-53 wherein the(−)-Ambrox has an odor threshold of from about 0.1 to about 0.5 ng/l.

Additional Aspects of the Invention

1. A squalene hopene cyclase (SHC)/homofarnesol Ambrox cyclase (HAC)derivative comprising an amino acid sequence having from 1-50 mutationsindependently selected from substitutions, deletions or insertionsrelative to SEQ ID No. 1.

2. The SHC/HAC derivative according to paragraph 1 wherein the SHCderivative comprises an amino acid sequence having from 1 to 40mutations, from 1-30 mutations, from 1-20 mutations, from 1-10 mutationsor from 1-6 mutations relative to SEQ ID No. 1.

3. The SHC/HAC derivative according to paragraph 1 or paragraph 2wherein the SHC/HAC derivative comprises an amino acid sequence havingat least 40% identity, at least 50% identity, or at least 60% identity,or at least 70% identity, or at least 80% identity, or at least 90%identity, or at least 95% identity, or at least 96% identity, or atleast 97% identity, or at least 98% identity, or at least 99% identityrelative to SEQ ID No. 1.

4. The SHC/HAC derivative according to paragraph 3 wherein the SHCvariant comprises an amino acid sequence having at least 95% identity toSEQ ID No. 1.

5. A SHC/HAC derivative comprising 1-10 mutations independently selectedfrom substitutions, deletions or insertions relative to SEQ ID No. 1wherein the one or more mutations other than an SHC active site mutationis/are located in domain 2 of the SHC enzyme (FIGS. 19 and/or 20).

6. The SHC/HAC derivative according to any one of paragraphs 1-5 whereinthe one or more mutations relative to SEQ ID No. 1 are selected fromTable 1 wherein if only one mutation is selected it is not F601Y.

7. The SHC/HAC derivative of paragraph 6 wherein at least 2, 3, 4, 5, 6,7, 8, 9 or 10 mutations are selected from Table 1 or Table 5.

8. The SHC/HAC derivative of paragraph 2 comprising an amino acidsequence that has up to 6 mutations relative to SEQ ID No. 1 andcomprises at least the substitutions F601Y or M132R in combination withat least any one or more of F129L and/or I432T.

9. The SHC/HAC derivative of paragraph 7 comprising an amino acidsequence which has up to 8 amino acid alterations relative to SEQ ID No.1 and comprises one or more than one amino acid alteration in a positionselected from the group consisting of positions 77, 129, 132, 192, 224,432, 579, 601 and 605 relative to SEQ ID No. 1 wherein the SHC/HACderivative has an increased HAC enzymatic activity relative to SEQ IDNo. 1.

10. The SHC/HAC derivative according to paragraph 9 comprising one ormore substitutions selected from the group consisting of: T77A, F129L,M132R, I92V, A224V, I432T, Q579H, F601Y and/or F605W relative to SEQ IDNo. 1.

11 The SHC/HAC derivative according to paragraph 10 comprising F601Y.

12. The SHC/HAC derivative according to paragraph 10 comprising F129L.

13. The SHC/HAC derivative according to paragraph 10 comprising F601Yand F129L.

14. The SHC/HAC derivative according to paragraph 10 comprising M132Rand I432T.

15. The SHC/HAC derivative according to paragraph 14 further comprisingthe amino acid substitution A224V.

16. The SHC/HAC derivative according to paragraph 14 further comprisingF601Y.

17. The SHC/HAC derivative according to paragraph 14 further comprisingF129L.

18. The SHC/HAC derivative according to paragraph 17 further comprisingF601Y.

19. The SHC/HAC derivative according to paragraph 11 further comprisingQ579H.

20. The SHC derivative according to paragraph 10 comprising T77A andI92V and F129L.

21. The SHC/HAC derivative according to any one of the precedingparagraphs having the amino acid sequence selected from the groupconsisting of SEQ ID No. 5, 7, 9, 11, 13, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39 and/or 171.

22. An isolated nucleotide sequence encoding the SHC derivativeaccording to any one of paragraphs 1-21.

23. The isolated nucleotide sequence according to paragraph 22 whereinthe nucleotide sequence is selected from the group consisting of SEQ IDNo. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40and/or 170.

24. A construct comprising the nucleotide sequence of paragraph 22 orparagraph 23.

25. A construct according to paragraph 24 comprising a promoterfunctionally linked to the nucleotide sequence of paragraph 22 or 23.

26. The construct of paragraph 25 wherein the promoter is an inducibleor a constitutive promoter.

27. A vector comprising the construct according to any one of paragraphs24-26.

28. The vector of paragraph 27 wherein the vector is a plasmid.

29. The vector according to paragraph 28 capable of directing expressionin host cells selected from prokaryotic, yeast, plant and insect hostcells.

30. The construct of any one of paragraphs 24-26 or the vector accordingto any one of paragraphs 27-29 wherein the construct or the vector iscapable of integration into the genome of a host cell selected fromprokaryotic, yeast, plant and insect host cells.

31. A recombinant host cell comprising a nucleotide sequence accordingto paragraph 22 or 23 or a construct according to any one of paragraphs24-26 or 30 or a vector according to any one of paragraphs 27-30.

32. The recombinant host cell according to paragraph 31 wherein the hostcell is selected from the group of prokaryotic host cells consisting ofthe bacteria of the genus Escherichia, Streptomyces, Bacillus,Pseudomonas, Lactobacillus and Lactococcus.

33. The recombinant host cell of paragraph 32 wherein the host cell isan E. coli host cell.

34. The recombinant host cell of paragraph 33 wherein the host celloverexpresses the gene encoding the SHC/HAC derivative.

35. A method of preparing the SHC/HAC derivative according to any one ofparagraphs 1-21 comprising the step of culturing one or more recombinanthost cells according to any one of paragraphs 31-34 under conditionswhich permit production of the SHC/HAC derivative enzyme.

36. The method of paragraph 35 wherein the cell culture takes placeunder conditions suitable for biocatalyst production.

37. A method of preparing (−)-Ambrox comprising converting homofarnesolto (−)-Ambrox using a recombinant host cell according to any one ofparagraphs 31-34 or by using a recombinant host cell comprising SEQ IDNo. 169 or SEQ ID No. 165 encoding a WT SHC/HAC wherein if a WT SHC/HACis used, the bioconversion of homofarnesol to (−)-Ambrox is carried outwith a solubilizing agent other than Triton X-100 or Taurodeoxycholate.

38. The method according to paragraph 37 wherein the conversion ofhomofarnesol to (−)-Ambrox under suitable bioconversion reactionconditions for the WT SHC/HAC or the SHC/HAC derivative enzyme.

39. The method according to paragraph 37 or 38 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place under suitable pH, temperature,solubilizing agent concentrations for the WT SHC/HAC or the SHC/HACderivative enzyme.

40. The method according to paragraph 39 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place at a temperature in the range offrom 30° C. to 60° C., at a pH in the range of about 4-8 and in thepresence of a solubilizing agent other than Triton X-100 orTaurodeoxycholate for the WT SHC/HAC enzyme.

41. The method according to any one of paragraphs 37-40 wherein theconversion of homofarnesol to (−)-Ambrox takes place using one or moreof the reaction conditions for the WT SHC/HAC or SHC/HAC derivativeenzyme as set out in Table 24 or Table 24a.

42. The method of any one of paragraphs 37-41 wherein the weight ratioof biocatalyst to homofarnesol is in the range of from about 0.5:1 to2:1 preferably about 1:1 or 0.5:1.

43. The method according to any one of paragraphs 37-42 wherein the cellgrowth and bioconversion reaction steps are carried out in the samereaction vessel.

44. The method according to any one of paragraphs 37-43 wherein thehomofarnesol substrate comprises one or more homofarnesol stereoisomers.

45. The method of paragraph 44 wherein the homofarnesol substratecomprises two homofarnesol stereoisomers.

46. The method of paragraph 45 wherein the homofarnesol substratecomprises EE:EZ stereoisomers.

47. The method according to any one of paragraphs 44-46 wherein thehomofarnesol comprises an EE:EZ stereoisomer mixture in the weightratios selected from the group consisting of: 100:00; 99:01; 98:02;97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12;87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19; 80:20; 79:21; 78:22;77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29 and 70:30.

48. The method of paragraph 47 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio is selected from the groupconsisting of: EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ69:31; and EE:EZ 66:34.

49. The method of paragraph 35 or 36 wherein the homofarnesol comprisesan EE:EZ stereoisomer mixture in a weight ratio of 80:20.

50. The method of any one of paragraphs 37-49 wherein (−)-Ambrox isproduced in admixture with one or more of the by-products (II), (IV)and/or (III).

51. The method of any one of paragraphs 37-50 wherein (−)-Ambrox isisolated from the bioconversion reaction mixture using an organicsolvent or a steam extraction/distillation step or (−)-Ambrox crystalsare isolated directly from the bioconversion reaction mixture by meansof filtration.

52. The method according to paragraph 51 wherein (−)-Ambrox is isolatedfrom the reaction mixture using an organic solvent.

53. The method of paragraph 52 wherein the (−)-Ambrox is selectivelycrystallized using an organic solvent.

54. The method of paragraph 52 or 53 wherein the (−)-Ambrox issubstantially free of the by-products (II), (IV) and/or (III).

55. (−)-Ambrox obtainable by the method of any one of paragraphs 51-54.

56. The (−)-Ambrox of paragraph 55 in a solid form, preferably in anamorphous or crystalline form.

57. A method for making a product containing (−)-Ambrox comprisingincorporating the (−)-Ambrox of paragraph 55 or 56 into the product,preferably a fragrance product, a cosmetic product, a cleaning product,a detergent product or a soap product.

58. A fragrance or cosmetic or a consumer care product comprising the(−)-Ambrox of paragraph 55 or 56.

59. A fragrance or cosmetic or consumer care composition comprising the(−)-Ambrox of paragraph 55 or 56 and one or more additional components.

60. The use of the (−)-Ambrox of paragraph 55 or 56 as part of afragrance or a cosmetic or a consumer product such as a fabric care,toiletry, beauty care and/or a cleaning product.

61. The use of a SHC/HAC derivative enzyme according to any one ofparagraphs 1-21, a nucleotide sequence according to paragraphs 22 or 23,a construct according to any one of paragraphs 24-26 or 30, a vectoraccording to any one of paragraphs 27-30 or a recombinant host cellaccording to any one of paragraphs 31-34 or a recombinant host cellexpressing a WT SHC/HAC for the bioconversion of homofarnesol to(−)-Ambrox wherein the WT SHC/HAC enzyme is used with a solubilizingagent other than Triton X-100 for the bioconversion reaction.

62. A process for preparing (−)-Ambrox or a stereoisomer mixture of(−)-Ambrox, wherein (3E,7E)-homofarnesol or a stereoisomer mixture of(3E,7E)-homofarnesol is converted enzymatically to give (−)-Ambrox or astereoisomer mixture of (−)-Ambrox wherein the enzymatic conversionusing an SHC/HAC enzyme is carried out under reaction conditionssuitable for the production of (−)-Ambrox and wherein if the reaction iscarried out in the presence of a solubilizing agent Triton X-100 is notused in combination with a WT SHC/HAC enzyme.

63. The process according to paragraph 62 wherein the process is carriedout using an SHC/HAC enzyme is selected from the group consisting ofAacSHC (SEQ ID No. 1), Zmo SHC1 (SEQ ID No. 2). ZmoSHC2, (SEQ ID No. 3);BjpSHC (SEQ ID No. 4), a SHC/HAC derivative selected from Table 1, Table5, Table 2, Table 6, Table 3, Table 7, Table 4 and/or Table 8, or asequence with at least 30% identity, at least 40% identity, at least 50%identity, or at least 60% identity, or at least 70% identity, or atleast 80% identity, or at least 90% identity, or at least 95% identity,or at least 96% identity, or at least 97% identity, or at least 98%identity, or at least 99% identity relative to SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, and/or SEQ ID No. 4.

64. The process according to paragraph 63 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place at a temperature in the range offrom 30° C. to 60° C., at a pH in the range of 4-8 and in the presenceof a solubilizing agent other than Triton X-100 for the WT SHC.

65. The process according to paragraph 64 wherein the reactionconditions for the WT SHC/HAC or each SHC/HAC derivative as set out inTable 24 or Table 24a are used.

66. The process according to any one of paragraphs 62-65 wherein theprocess comprises (a) culturing one or more recombinant host cellsexpressing a WT SHC or SHC derivative enzyme under conditions whichpermit expression of the WT SHC or SHC/HAC derivative polypeptide priorto the conversion of E,E-homofarnesol to (−)-Ambrox.

67. The process according to paragraph 66 wherein the culturing step andsubsequence conversion step takes place in the same reaction vesselunder different reaction conditions.

68. The process according to paragraph 67 wherein the culturing step isat a pH range of about 6 to about 7 and the homofarnesol to (−)-Ambroxstep is at a pH range of about 4.8-5.5.

69. The process according to any one of paragraphs 62-68 wherein thehomofarnesol substrate comprises EE: EZ stereoisomers.

70. The process according to paragraph 69 wherein the homofarnesolcomprises an EE:EZ stereoisomer mixture in a weight ratio is selectedfrom the group consisting of: EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14;EE:EZ 70:30; EE:EZ 69:31; and EE:EZ 66:34.

71. The process of paragraph 70 wherein the homofarnesol comprises EE:EZin a weight ratio of 80:20.

72. The method of any one of paragraphs 62-71 wherein (−)-Ambrox isproduced in admixture with one or more of the by-products (II), (IV)and/or (III).

73. The method of any one of paragraphs 62-72 wherein (−)-Ambrox isisolated from the reaction mixture using an organic solvent or a steamextraction/distillation step or filtration.

74. The method according to paragraph 73 wherein (−)-Ambrox is isolatedfrom the reaction mixture using an organic solvent.

75. The method of paragraph 74 wherein (−)-Ambrox is selectivelycrystallized using an organic solvent.

76. The method of paragraph 74 or 75 wherein the (−)-Ambrox issubstantially free of the by-products (II), (IV) and/or (III).

77. (−)-Ambrox obtainable by the method of any one of paragraphs 72-76.

78. The (−)-Ambrox of paragraph 26 in a solid form, preferably in anamorphous or crystalline form.

79. A method for making a product comprising incorporating the(−)-Ambrox of paragraphs 77 or 78 into the product.

80. The method of paragraph 79 wherein the product is a fragranceproduct, a cosmetic product, a cleaning product, a detergent product ora soap product.

81. A fragrance or cosmetic or a consumer care product comprising the(−)-Ambrox of paragraph 77 or 78.

82. A fragrance or cosmetic or consumer care composition comprising the(−)-Ambrox of paragraph 77 or 78 and an additional component.

83. The use of the (−)-Ambrox of paragraph 77 or 78 as part of afragrance or cosmetic consumer care product.

Additional Aspects of the Invention (ZmoSHC1)

1. A squalene hopene cyclase (SHC)/homofarnesol Ambrox cyclase (HAC)derivative comprising an amino acid sequence having from 1-50 mutationsindependently selected from substitutions, deletions or insertionsrelative to SEQ ID No. 2.

2. The SHC/HAC derivative according to paragraph 1 wherein the SHCderivative comprises an amino acid sequence having from 1 to 40mutations, from 1-30 mutations, from 1-20 mutations, from 1-10 mutationsor from 1-6 mutations relative to SEQ ID No. 2.

3. The SHC/HAC derivative according to paragraph 1 or paragraph 2wherein the SHC/HAC derivative comprises an amino acid sequence havingat least 40% identity, at least 50% identity, or at least 60% identity,or at least 70% identity, or at least 80% identity, or at least 90%identity, or at least 95% identity, or at least 96% identity, or atleast 97% identity, or at least 98% identity, or at least 99% identityrelative to SEQ ID No. 2.

4. The SHC/HAC derivative according to paragraph 3 wherein the SHCvariant comprises an amino acid sequence having at least 95% identity toSEQ ID No. 2.

5. A SHC/HAC derivative comprising 1-10 mutations independently selectedfrom substitutions, deletions or insertions relative to SEQ ID No. 2wherein the one or more mutations other than an SHC active site mutationis/are located in domain 2 of the SHC enzyme (FIGS. 19 and/or 20).

6. The SHC/HAC derivative according to any one of paragraphs 1-5 whereinthe one or more mutations relative to SEQ ID No. 2 are selected fromTable 2 wherein if only one mutation is selected it is not F668Y.

7. The SHC/HAC derivative of paragraph 6 wherein at least 2, 3, 4, 5, 6,7, 8, 9 or 10 mutations are selected from Table 2 and/or Table 6.

8. The SHC/HAC derivative of paragraph 2 comprising an amino acidsequence that has up to 6 mutations relative to SEQ ID No 2 andcomprises at least the substitutions F668Y or Y185R in combination withat least any one or more of F182L and/or I498T.

9. The SHC/HAC derivative of paragraph 7 comprising an amino acidsequence which has up to 8 amino acid alterations relative to SEQ ID No.2 and comprises one or more one amino acid alteration in a positionselected from the group consisting of positions 129, 145, 182, 185, 282,498, 647 and 668 relative to SEQ ID No. 2 wherein the SHC/HAC derivativehas an increased HAC enzymatic activity relative to SEQ ID No. 2.

10. The SHC/HAC derivative according to paragraph 9 comprising one ormore substitutions selected from the group consisting of S129A, V145V,F182L, Y185R, G282V, I498T, H646H and F668Y relative to SEQ ID No. 2.

11. The SHC/HAC derivative according to paragraph 10 comprising F668Y.

12. The SHC/HAC derivative according to paragraph 10 comprising F182L.

13. The SHC/HAC derivative according to paragraph 10 comprising F668Yand F182L.

14. The SHC/HAC derivative according to paragraph 10 comprising Y185Rand I498T.

15. The SHC/HAC derivative according to paragraph 14 further comprisingG282V.

16. The SHC/HAC derivative according to paragraph 14 further comprisingF668Y.

17. The SHC/HAC derivative according to paragraph 14 further comprisingF182L.

18. The SHC/HAC derivative according to paragraph 17 further comprisingF668Y.

19. The SHC/HAC derivative according to paragraph 11 further comprisingH646H.

20. The SHC derivative according to paragraph 10 comprising S129A andV145V and F182L.

21. The SHC/HAC derivative according to any one of the precedingparagraphs having the amino acid sequence selected from the groupconsisting of SEQ ID No. 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73 and/or 75.

22. An isolated nucleotide sequence encoding the SHC derivativeaccording to any one of paragraphs 1-21.

23. The isolated nucleotide sequence according to paragraph 22 whereinthe nucleotide sequence is selected from the group consisting of SEQ IDNo. 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74and/or 76.

24. A construct comprising the nucleotide sequence of paragraph 22 orparagraph 23.

25. A construct according to paragraph 24 comprising a promoterfunctionally linked to the nucleotide sequence of paragraph 22 or 23.

26. The construct of paragraph 25 wherein the promoter is an inducibleor a constitutive promoter.

27. A vector comprising the construct according to any one of paragraphs24-26.

28. The vector of paragraph 27 wherein the vector is a plasmid.

29. The vector according to paragraph 28 capable of directing expressionin host cells selected from prokaryotic, yeast, plant and insect hostcells.

30. The construct of any one of paragraphs 24-26 or the vector accordingto any one of paragraphs 27-29 wherein the construct or the vector iscapable of integration into the genome of a host cell selected fromprokaryotic, yeast, plant and insect host cells.

31. A recombinant host cell comprising a nucleotide sequence accordingto paragraph 22 or 23 or a construct according to any one of paragraphs24-26 or 30 or a vector according to any one of paragraphs 27-30.

32. The recombinant host cell according to paragraph 31 wherein the hostcell is selected from the group of prokaryotic host cells consisting ofthe bacteria of the genus Escherichia, Streptomyces, Bacillus,Pseudomonas, Lactobacillus and Lactococcus.

33. The recombinant host cell of paragraph 32 wherein the host cell isan E. coli host cell.

34. The recombinant host cell of paragraph 33 wherein the host celloverexpresses the gene encoding the SHC/HAC derivative.

35. A method of preparing the SHC/HAC derivative according to any one ofparagraphs 1-21 comprising the step of culturing one or more recombinanthost cells according to any one of paragraphs 31-34 under conditionswhich permit production of the SHC/HAC derivative enzyme.

36. The method of paragraph 35 wherein the cell culture takes placeunder conditions suitable for biocatalyst production.

37. A method of preparing (−)-Ambrox comprising converting homofarnesolto (−)-Ambrox using a recombinant host cell according to any one ofparagraphs 31-34 or by using a recombinant host cell comprising SEQ IDNo. 166 encoding a WT SHC/HAC wherein if a WT SHC/HAC is used, thebioconversion of homofarnesol to (−)-Ambrox is carried out with asolubilizing agent other than Triton X-100.

38. The method according to paragraph 37 wherein the conversion ofhomofarnesol to (−)-Ambrox under suitable bioconversion reactionconditions for the WT SHC/HAC or the SHC/HAC derivative enzyme.

39. The method according to paragraph 37 or 38 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place under suitable pH, temperature,solubilizing agent concentrations for the WT SHC/HAC or the SHC/HACderivative enzyme.

40. The method according to paragraph 39 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place at a temperature in the range offrom 30° C. (to 60° C., at a pH in the range of about 4-8 and in thepresence of a solubilizing agent other than Triton X-100 for the WTSHC/HAC enzyme.

41. The method according to any one of paragraphs 37-40 wherein theconversion of homofarnesol to (−)-Ambrox takes place using one or moreof the reaction conditions for the WT SHC/HAC or SHC/HAC derivativeenzyme as set out in Table 24 or Table 24a.

42. The method of any one of paragraphs 37-41 wherein the weight ratioof biocatalyst to homofarnesol is in the range of from about 0.5:1 to2:1, preferably about 1:1 or 0.5:1.

43. The method according to any one of paragraphs 37-42 wherein the cellgrowth and bioconversion reaction steps are carried out in the samereaction vessel.

44. The method according to any one of paragraphs 37-43 wherein thehomofarnesol substrate comprises one or more homofarnesol stereoisomers.

45. The method of paragraph 44 wherein the homofarnesol substratecomprises two homofarnesol stereoisomers.

46. The method of paragraph 45 wherein the homofarnesol substratecomprises EE:EZ stereoisomers.

47. The method according to any one of paragraphs 44-46 wherein thehomofarnesol comprises an EE:EZ stereoisomer mixture in the weightratios selected from the group consisting of 100:00; 99:01; 98:02;97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12;87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19; 80:20; 79:21; 78:22;77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29 and 70:30.

48. The method of paragraph 47 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio is selected from the groupconsisting of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ69:31; and EE:EZ 66:34.

49. The method paragraph 35 or 36 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio of 80:20.

50. The method of any one of paragraphs 37-49 wherein (−)-Ambrox isproduced in admixture with one or more of the by-products (II), (IV)and/or (III).

51. The method of any one of paragraphs 37-50 wherein (−)-Ambrox isisolated from the bioconversion reaction mixture using an organicsolvent or a steam extraction/distillation step, or filtration.

52. The method according to paragraph 51 wherein (−)-Ambrox is isolatedfrom the reaction mixture using an organic solvent.

53. The method of paragraph 52 wherein the (−)-Ambrox is selectivelycrystallized from (−)-Ambrox using an organic solvent.

54. The method of paragraph 52 or 53 wherein the (−)-Ambrox issubstantially free of the by-products (II), (IV) and/or (III).

55. (−)-Ambrox obtainable by the method of any one of paragraphs 51-54.

56. The (−)-Ambrox of paragraph 55 in a solid form, preferably in anamorphous or crystalline form.

57. A method for making a product containing (−)-Ambrox comprisingincorporating the (−)-Ambrox of paragraph 55 or 56 into the product,preferably a fragrance product, a cosmetic product, a cleaning product,a detergent product or a soap product.

58. A fragrance or cosmetic or a consumer care product comprising the(−)-Ambrox of paragraph 55 or 56.

59. A fragrance or cosmetic or consumer care composition comprising the(−)-Ambrox of paragraph 55 or 56 and one or more additional components.

60. The use of the (−)-Ambrox of paragraph 55 or 56 as part of afragrance or a cosmetic or a consumer product such as a fabric care,toiletry, beauty care and/or a cleaning product.

61. The use of a SHC/HAC derivative enzyme according to any one ofparagraphs 1-21, a nucleotide sequence according to paragraphs 22 or 23,a construct according to any one of paragraphs 24-26 or 30, a vectoraccording to any one of paragraphs 27-30 or a recombinant host cellaccording to any one of paragraphs 31-34 or a recombinant host cellexpressing a WT SHC/HAC for the bioconversion of homofarnesol to(−)-Ambrox wherein the WT SHC/HAC enzyme is used with a solubilizingagent other than Triton X-100 for the bioconversion reaction.

Further Aspects of the Invention (ZmoSHC2)

1. A squalene hopene cyclase (SHC) homofarnesol Ambrox cyclase (HAC)derivative comprising an amino acid sequence having from 1-50 mutationsindependently selected from substitutions, deletions or insertionsrelative to SEQ ID No. 3.

2. The SHC/HAC derivative according to paragraph 1 wherein the SHCderivative comprises an amino acid sequence having from 1 to 40mutations, from 1-30 mutations, from 1-20 mutations, from 1-10 mutationsor from 1-6 mutations relative to SEQ ID No. 3.

3. The SHC/HAC derivative according to paragraph 1 or paragraph 2wherein the SHC/HAC derivative comprises an amino acid sequence havingat least 40% identity, at least 50% identity, or at least 60% identity,or at least 70% identity, or at least 80% identity, or at least 90%identity, or at least 95% identity, or at least 96% identity, or atleast 97% identity, or at least 98% identity, or at least 99% identityrelative to SEQ ID No. 3.

4. The SHC/HAC derivative according to paragraph 3 wherein the SHCvariant comprises an amino acid sequence having at least 95% identity toSEQ ID No. 3.

5. A SHC/HAC derivative comprising 1-10 mutations independently selectedfrom substitutions, deletions or insertions relative to SEQ ID No. 3wherein the one or more mutations other than n SHC active site mutationis/are located in domain 2 of the SHC enzyme (FIGS. 19 and/or 20).

6. The SHC/HAC derivative according to any one of paragraphs 1-5 whereinthe one or more mutations relative to SEQ ID No. 3 are selected fromTable 3 wherein if only one mutation is selected it is not F620Y.

7. The SHC/HAC derivative of paragraph 6 wherein at least 2, 3, 4, 5, 6,7, 8, 9 or 10 mutations are selected from Table 3 and/or Table 7.

8. The SHC/HAC derivative of paragraph 2 comprising an amino acidsequence that has up to 6 mutations relative to SEQ ID No. 3 andcomprises at least the substitutions F620Y or I140R in combination withat least any one or more of F137L and/or I450T.

9. The SHC/f-AC derivative of paragraph 7 comprising an amino acidsequence which has up to 8 amino acid alterations relative to SEQ ID No.3 and comprises one or more one amino acid alteration in a positionselected from the group consisting of positions 85, 100, 137, 140, 233,450, 598 and 620 relative to SEQ ID No. 3 wherein the SHC/HAC derivativehas an increased HAC enzymatic activity relative to SEQ ID No. 3.

10. The SHC/HAC derivative according to paragraph 9 comprising one ormore substitutions selected from the group consisting of: G85A, V100V,F137L, I140R V233V, I450T, N598H and F620Y relative to SEQ ID No. 3.

11. The SHC/HAC derivative according to paragraph 10 comprising F620Y.

12. The SHC/HAC derivative according to paragraph 10 comprising F137L.

13. The SHC/HAC derivative according to paragraph 10 comprising F620Yand F137L.

14. The SHC/HAC derivative according to paragraph 10 comprising I140Rand I450T.

15. The SHC/HAC derivative according to paragraph 14 further comprisingV233V.

16. The SHC/HAC derivative according to paragraph 14 further comprisingF620Y.

17. The SHC/HAC derivative according to paragraph 14 further comprisingF137L.

18. The SHC/HAC derivative according to paragraph 17 further comprisingF620Y.

19. The SHC/HAC derivative according to paragraph 11 further comprisingN598H.

20. The SHC derivative according to paragraph 10 comprising G85A andV100V and F137L.

21. The SHC/HAC derivative according to any one of the precedingparagraphs having the amino acid sequence selected from the groupconsisting of SEQ ID No. 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109 and/or 111.

22. An isolated nucleotide sequence encoding the SHC derivativeaccording to any one of paragraphs 1-21.

23. The isolated nucleotide sequence according to paragraph 22 whereinthe nucleotide sequence is selected from the group consisting of SEQ IDNo. 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110 and/or 112.

24. A construct comprising the nucleotide sequence of paragraph 22 orparagraph 23.

25. A construct according to paragraph 24 comprising a promoterfunctionally linked to the nucleotide sequence of paragraph 22 or 23.

26. The construct of paragraph 25 wherein the promoter is an inducibleor a constitutive promoter.

27. A vector comprising the construct according to any one of paragraphs24-26.

28. The vector of paragraph 27 wherein the vector is a plasmid.

29. The vector according to paragraph 28 capable of directing expressionin host cells selected from prokaryotic, yeast, plant and insect hostcells.

30. The construct of any one of paragraphs 24-26 or the vector accordingto any one of paragraphs 27-29 wherein the construct or the vector iscapable of integration into the genome of a host cell selected fromprokaryotic, yeast, plant and insect host cells.

31. A recombinant host cell comprising a nucleotide sequence accordingto paragraph 22 or 23 or a construct according to any one of paragraphs24-26 or 30 or a vector according to any one of paragraphs 27-30.

32. The recombinant host cell according to paragraph 31 wherein the hostcell is selected from the group of prokaryotic host cells consisting ofthe bacteria of the genus Escherichia, Streptomyces, Bacillus,Pseudomonas, Lactobacillus and Lactococcus.

33. The recombinant host cell of paragraph 32 wherein the host cell isan E. coli host cell.

34. The recombinant host cell of paragraph 33 wherein the host celloverexpresses the gene encoding the SHC/HAC derivative.

35. A method of preparing the SHC/HAC derivative according to any one ofparagraphs 1-21 comprising the steps of: (a) culturing one or morerecombinant host cells according to any one of paragraphs 31-34 underconditions which permit production of the SHC/HAC derivative enzyme.

36. The method of paragraph 35 wherein the cell culture takes placeunder conditions suitable for biocatalyst production.

37. A method of preparing (−)-Ambrox comprising converting homofarnesolto (−)-Ambrox using a recombinant host cell according to any one ofparagraphs 31-34 or by using a recombinant host cell comprising SEQ IDNo. 167 encoding a WT SHC/HAC wherein if a WT SHC/HAC is used, thebioconversion of homofarnesol to (−)-Ambrox is carried out with asolubilizing agent other than Triton X-100.

38. The method according to paragraph 37 wherein the conversion ofhomofarnesol to (−)-Ambrox under suitable bioconversion reactionconditions for the WT SHC/HAC or the SHC/HAC derivative enzyme.

39. The method according to paragraph 37 or 38 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place under suitable pH, temperature,solubilizing agent concentrations for the WT SHC/HAC or the SHC/HACderivative enzyme.

40. The method according to paragraph 39 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place at a temperature in the range offrom 30° C. to 60° C., at a pH in the range of about 4-8 and in thepresence of a solubilizing agent other than Triton X-100 for the WTSHC/HAC enzyme.

41. The method according to any one of paragraphs 37-40 wherein theconversion of homofarnesol to (−)-Ambrox takes place using one or moreof the reaction conditions for the WT SHC/HAC or SHC/HAC derivativeenzyme as set out in Table 24 or Table 24a.

42. The method of any one of paragraphs 37-41 wherein the weight ratioof biocatalyst to homofarnesol is in the range of from about 0.5:1 to2:1, preferably about 1:1 or 0.5:1.

43. The method according to any one of paragraphs 37-42 wherein the cellgrowth and bioconversion reaction steps are carried out in the samereaction vessel.

44. The method according to any one of paragraphs 37-43 wherein thehomofarnesol substrate comprises one or more homofarnesol stereoisomers.

45. The method of paragraph 44 wherein the homofarnesol substratecomprises two homofarnesol stereoisomers.

46. The method of paragraph 45 wherein the homofarnesol substratecomprises EE:EZ stereoisomers.

47. The method according to any one of paragraphs 44-46 wherein thehomofarnesol comprises an EE:EZ stereoisomer mixture in the weightratios selected from the group consisting of: 100:00; 99:01; 98:02;97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12;87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19; 80:20; 79:21; 78:22;77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29 and 70:30.

48. The method of paragraph 47 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio is selected from the groupconsisting of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ69:31; and EE:EZ 66:34.

49. The method paragraph 35 or 36 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio of 80:20.

50. The method of any one of paragraphs 37-49 wherein (−)-Ambrox isproduced in admixture with one or more of the by-products (II), (IV)and/or (III).

51. The method of any one of paragraphs 37-50 wherein (−)-Ambrox isisolated from the bioconversion reaction mixture using an organicsolvent or a steam extraction/distillation step, or filtration.

52. The method according to paragraph 51 wherein (−)-Ambrox is isolatedfrom the reaction mixture using an organic solvent.

53. The method of paragraph 52 wherein the (−)-Ambrox is selectivelycrystallized from (−)-Ambrox using an organic solvent.

54. The method of paragraph 52 or 53 wherein the (−)-Ambrox issubstantially free of the by-products (II), (IV) and/or (III).

55. (−)-Ambrox obtainable by the method of any one of paragraphs 51-54.

56. The (−)-Ambrox of paragraph 55 in a solid form, preferably in anamorphous or crystalline form.

57. A method for making a product containing (−)-Ambrox comprisingincorporating the (−)-Ambrox of paragraph 55 or 56 into the product,preferably a fragrance product, a cosmetic product, a cleaning product,a detergent product or a soap product.

58. A fragrance or cosmetic or a consumer care product comprising the(−)-Ambrox of paragraph 55 or 56.

59. A fragrance or cosmetic or consumer care composition comprising the(−)-Ambrox of paragraph 55 or 56 and one or more additional components.

60. The use of the (−)-Ambrox of paragraph 55 or 56 as part of afragrance or a cosmetic or a consumer product such as a fabric care,toiletry, beauty care and/or a cleaning product.

61. The use of a SHC/HAC derivative enzyme according to any one ofparagraphs 1-21, a nucleotide sequence according to paragraphs 22 or 23,a construct according to any one of paragraphs 24-26 or 30, a vectoraccording to any one of paragraphs 27-30 or a recombinant host cellaccording to any one of paragraphs 31-34 or a recombinant host cellexpressing a WT SHC/HAC for the bioconversion of homofarnesol to(−)-Ambrox wherein the WT SHC/HAC enzyme is used with a solubilizingagent other than Triton X-100 for the bioconversion reaction.

Additional Aspects of the Invention (BJpSHC)

1. A squalene hopene cyclase (SHC)/homofarnesol Ambrox cyclase (HAC)derivative comprising an amino acid sequence having from 1-50 mutationsindependently selected from substitutions, deletions or insertionsrelative to SEQ ID No. 4.

2. The SHC/HAC derivative according to paragraph 1 wherein the SHCderivative comprises an amino acid sequence having from 1 to 40mutations, from 1-30 mutations, from 1-20 mutations, from 1-10 mutationsor from 1-6 mutations relative to SEQ ID No. 4.

3. The SHC/HAC derivative according to paragraph 1 or paragraph 2wherein the SHC/HAC derivative comprises an amino acid sequence havingat least 40% identity, at least 50% identity, or at least 60% identity,or at least 70% identity, or at least 80% identity, or at least 90%identity, or at least 95% identity, or at least 96% identity, or atleast 97% identity, or at least 98% identity, or at least 99% identityrelative to SEQ ID No. 4.

4. The SHC/HAC derivative according to paragraph 3 wherein the SHCvariant comprises an amino acid sequence having at least 95% identity toSEQ ID No. 4.

5. A SHC/HAC derivative comprising 1-10 mutations independently selectedfrom substitutions, deletions or insertions relative to SEQ ID No. 4wherein the one or more mutations other than an SHC active site mutationis/are located in domain 2 of the SHC enzyme (FIGS. 19 and/or 20).

6. The SHC/HAC derivative according to any one of paragraphs 1-5 whereinthe one or more mutations relative to SEQ ID No. 4 are selected fromTable 4 wherein if only one mutation is selected it is not F628Y.

7. The SHC/HAC derivative of paragraph 6 wherein at least 2, 3, 4, 5, 6,7, 8, 9 or 10 mutations are selected from Table 4 and/or Table 8.

8. The SHC/HAC derivative of paragraph 2 comprising an amino acidsequence that has up to 6 mutations relative to SEQ ID No. 4 andcomprises at least the substitutions F628Y or I140R in combination withat least any one or more of F137L and/or I450T.

9. The SHC/HAC derivative of paragraph 7 comprising an amino acidsequence which has up to 8 amino acid alterations relative to SEQ ID No.4 and comprises one or more one amino acid alteration in a positionselected from the group consisting of positions 88, 104, 141, 144, 241,459, 607 and 628 relative to SEQ ID No. 4 wherein the SHC/HAC derivativehas an increased HAC enzymatic activity relative to SEQ ID No. 4.

10. The SHC/HAC derivative according to paragraph 9 comprising one ormore substitutions selected from the group consisting of A88A, V104V,F141L, Y144R, V241V, I459T, M607H and F628Y relative to SEQ ID No. 4.

11. The SHC/HAC derivative according to paragraph 10 comprising F628Y.

12. The SHC/HAC derivative according to paragraph 10 comprising F141 L.

13. The SHC/HAC derivative according to paragraph 10 comprising F628Yand F41L.

14. The SHC/HAC derivative according to paragraph 10 comprising Y144Rand I459T.

15. The SHC/HAC derivative according to paragraph 14 further comprisingV241V.

16. The SHC/HAC derivative according to paragraph 14 further comprisingF628Y.

17. The SHC/HAC derivative according to paragraph 14 further comprisingF141L.

18. The SHC/HAC derivative according to paragraph 17 further comprisingF628Y.

19. The SHC/HAC derivative according to paragraph 11 further comprisingM607H.

20. The SHC derivative according to paragraph 10 comprising S129A andV145V and F182L.

21. The SHC/HAC derivative according to any one of the precedingparagraphs having the amino acid sequence selected from the groupconsisting of SEQ ID No. 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145 and/or 147.

22. An isolated nucleotide sequence encoding the SHC derivativeaccording to any one of paragraphs 1-21.

23. The isolated nucleotide sequence according to paragraph 22 whereinthe nucleotide sequence is selected from the group consisting of SEQ IDNo. 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146 and/or 148.

24. A construct comprising the nucleotide sequence of paragraph 22 orparagraph 23.

25. A construct according to paragraph 24 comprising a promoterfunctionally linked to the nucleotide sequence of paragraph 22 or 23.

26. The construct of paragraph 25 wherein the promoter is an inducibleor a constitutive promoter.

27. A vector comprising the construct according to any one of paragraphs24-26.

28. The vector of paragraph 27 wherein the vector is a plasmid.

29. The vector according to paragraph 28 capable of directing expressionin host cells selected from prokaryotic, yeast, plant and insect hostcells.

30. The construct of any one of paragraphs 24-26 or the vector accordingto any one of paragraphs 27-29 wherein the construct or the vector iscapable of integration into the genome of a host cell selected fromprokaryotic, yeast, plant and insect host cells.

31. A recombinant host cell comprising a nucleotide sequence accordingto paragraph 22 or 23 or a construct according to any one of paragraphs24-26 or 30 or a vector according to any one of paragraphs 27-30.

32. The recombinant host cell according to paragraph 31 wherein the hostcell is selected from the group of prokaryotic host cells consisting ofthe bacteria of the genus Escherichia, Streptomyces, Bacillus,Pseudomonas, Lactobacillus and Lactococcus.

33. The recombinant host cell of paragraph 32 wherein the host cell isan E. coli host cell.

34. The recombinant host cell of paragraph 33 wherein the host celloverexpresses the gene encoding the SHC/HAC derivative.

35. A method of preparing the SHC/HAC derivative according to any one ofparagraphs 1-21 comprising the steps of: (a) culturing one or morerecombinant host cells according to any one of paragraphs 31-34 underconditions which permit production of the SHC/HAC derivative enzyme.

36. The method of paragraph 35 wherein the cell culture takes placeunder conditions suitable for biocatalyst production.

37. A method of preparing (−)-Ambrox comprising converting homofarnesolto (−)-Ambrox using a recombinant host cell according to any one ofparagraphs 31-34 or by using a recombinant host cell comprising SEQ IDNo. 168 encoding a WT SHC/HAC wherein if a WT SHC/HAC is used, thebioconversion of homofarnesol to (−)-Ambrox is carried out with asolubilising agent other than Triton X-100.

38. The method according to paragraph 37 wherein the conversion ofhomofarnesol to (−)-Ambrox under suitable bioconversion reactionconditions for the WT SHC/HAC or the SHC/HAC derivative enzyme.

39. The method according to paragraph 37 or 38 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place under suitable pH, temperature,solubilising agent concentrations for the WT SHC/HAC or the SHC/HACderivative enzyme.

40. The method according to paragraph 39 wherein the conversion ofhomofarnesol to (−)-Ambrox takes place at a temperature in the range offrom 30° C. to 60° C., at a pH in the range of about 4-8 and in thepresence of a solubilising agent other than Triton X-100 for the WTSHC/HAC enzyme.

41. The method according to any one of paragraphs 37-40 wherein theconversion of homofarnesol to (−)-Ambrox takes place using one or moreof the reaction conditions for the WT SHC/HAC or SHC/HAC derivativeenzyme as set out in Table 24 or Table 24a.

42. The method of any one of paragraphs 37-41 wherein the weight ratioof biocatalyst to homofarnesol is in the range of from about 0.5:1 to2:1, preferably about 1:1 or 0.5:1.

43. The method according to any one of paragraphs 37-42 wherein the cellgrowth and bioconversion reaction steps are carried out in the samereaction vessel.

44. The method according to any one of paragraphs 27-31 wherein thehomofarnesol substrate comprises one or more homofarnesol stereoisomers.

45. The method of paragraph 44 wherein the homofarnesol substratecomprises two homofarnesol stereoisomers.

46. The method of paragraph 45 wherein the homofarnesol substratecomprises EE:EZ stereoisomers.

47. The method according to any one of paragraphs 44-46 wherein thehomofarnesol comprises an EE:EZ stereoisomer mixture in the weightratios selected from the group consisting of 100:00; 99:01; 98:02;97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12;87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19; 80:20; 79:21; 78:22;77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29 and 70:30.

48. The method of paragraph 47 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio is selected from the groupconsisting of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ69:31; and EE:EZ 66:34.

49. The method paragraph 35 or 36 wherein the homofarnesol comprises anEE:EZ stereoisomer mixture in a weight ratio of 80:20.

50. The method of any one of paragraphs 37-49 wherein (−)-Ambrox isproduced in admixture with one or more of the by-products (II), (IV)and/or (III).

51. The method of any one of paragraphs 37-50 wherein (−)-Ambrox isisolated from the bioconversion reaction mixture using an organicsolvent or a steam extraction/distillation step, or filtration.

52. The method according to paragraph 51 wherein (−)-Ambrox is isolatedfrom the reaction mixture using an organic solvent.

53. The method of paragraph 52 wherein the (−)-Ambrox is selectivelycrystallized from (−)-Ambrox using an organic solvent.

54. The method of paragraph 52 or 53 wherein the (−)-Ambrox issubstantially free of the by-products (II). (IV) and/or (III). 55.(−)-Ambrox obtainable by the method of any one of paragraphs 51-54.

56. The (−)-Ambrox of paragraph 55 in a solid form, preferably in anamorphous or crystalline form.

57. A method for making a product containing (−)-Ambrox comprisingincorporating the (−)-Ambrox of paragraph 55 or 56 into the product,preferably a fragrance product, a cosmetic product, a cleaning product,a detergent product or a soap product.

58. A fragrance or cosmetic or a consumer care product comprising the(−)-Ambrox of paragraph 55 or 56.

59. A fragrance or cosmetic or consumer care composition comprising the(−)-Ambrox of paragraph 55 or 56 and one or more additional components.

60. The use of the (−)-Ambrox of paragraph 55 or 56 as part of afragrance or a cosmetic or a consumer product such as a fabric care,toiletry, beauty care and/or a cleaning product.

61. The use of a SHC/HAC derivative enzyme according to any one ofparagraphs 1-21, a nucleotide sequence according to paragraphs 22 or 23,a construct according to any one of paragraphs 24-26 or 30, a vectoraccording to any one of paragraphs 27-30 or a recombinant host cellaccording to any one of paragraphs 31-34 or a recombinant host cellexpressing a WT SHC/HAC for the bioconversion of homofarnesol to(−)-Ambrox wherein the WT SHC/HAC enzyme is used with a solubilizingagent other than Triton X-100 for the bioconversion reaction.

In another aspect, there is provided an SHC crystal model structure(CMS) based on the structural coordinates of SHC with an amino acidsequence of SHC or derivative described herein. The SHC CMS comprises asqualene/homofarnesol binding pocket domain (SHBD) that comprises asqualene/homofarnesol binding pocket (SHBP) and a squalene/homofarnesolsubstrate bound to the SBD (eg. see FIGS. 19 and 20). This SHC crystalmodel structure (CMS) facilitates in-silico testing of potential SHC/HACderivative enzyme candidates.

Thus, in still other embodiments, the present disclosure provides amethod of screening for an enzyme (eg. a SHC/HAC derivative) capable ofbinding to a SHBD wherein the method comprises the use of the SHC/HACCMS. In another aspect, the present disclosure provides a method forscreening for an enzyme (eg. a reference SHC or a SHC/HAC derivative)capable of binding to the SHBP, and the method comprises contacting theSHBP with a test compound (eg. a SHC derivative), and determining ifsaid test compound binds to said SHBP. In some embodiments, the methodis to screen for a test compound (eg. a modulator) useful in modulatingthe activity of an SHC derivative enzyme.

In another aspect, the present disclosure provides a method forpredicting, simulating or modelling the molecular characteristics and/ormolecular interactions of a reference SHC and/or a SHC/HAC derivativewith a squalene/homofarnesol binding domain (SHBD) comprising the use ofa computer model, said computer model comprising, using or depicting thestructural coordinates of a squalene/homofarnesol binding domain asdefined above to provide an image of said ligand binding domain and tooptionally display said image.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Theterm “comprising” also means “including” as well as “consisting” eg. acomposition “comprising” X may consist exclusively of X or may includesomething additional eg. X+Y. It must be noted also that, as used inthis specification and the appended claims, the singular forms “a”, “an”and “the” include plural referents unless the content clearly dictatesotherwise. By way of example, a reference to “a gene” or “an enzyme” isa reference to “one or more genes” or “one or more enzymes”.

It is to be understood that this disclosure is not limited to theparticular methodology, protocols and reagents described herein as thesemay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present disclosure which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by the person skilled in the art. In accordance withthe present disclosure there may be conventional molecular biology,microbiology, and recombinant DNA techniques employed which are withinthe skill of the art.

This disclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The disclosure iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. nd Kolbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, GenBank Accession Number sequence submissions etc.),whether supra or infra, is hereby incorporated by reference in itsentirety.

The examples described herein are illustrative of the present disclosureand are not intended to be limitations thereon. Different embodiments ofthe present disclosure have been described according to the presentdisclosure. Many modifications and variations may be made to thetechniques described and illustrated herein without departing from thespirit and scope of the disclosure. Accordingly, it should be understoodthat the examples are illustrative only and are not limiting upon thescope of the disclosure.

TABLE 10 provides the accession number for AacSHC SHC source strain (SHCname) Reference Accession No. Alicyclobacillus JP2009-060799 (Kao)NBRC15652 acidocaldarius Neumann et al Bio1 Chem (WT AacSHC) (1986) 367;723-729

TABLE 11 provides accession number for ZmoSHC SEQ ID No. SHC sourceaccording strain Strain and to WO 2010139719 (SHC name) ReferenceAccession No. US2012/0135477 Zymomonas WO2010139719 ATCC31821 SEQ ID No.1 mobilis US2012/0135477 PF62207_2 SEQ ID No. 2 (WT ZmoSHC) Genpept.Accession No AAV90172 Reipen et al (1995) EMBL/Genbank Microbiology141:155-161. Accession No. for X80766

TABLE 12 shows sources of other SHC enzymes from WO2010139719 SEQ In No.according to SHC source strain WO 2010139719 (SHC name) ReferenceAccession No. US2012/0135477 Bradryhizobium WO2010139719 PF62207_5 SEQID No. 5 japonicum US2012/0135477 (WT BjpSHC) Burkholderia WO2010139719SEQ ID No. 6 ambifaria US2012/0135477 Burkholderia WO2010139719 SEQ IDNo. 7 ambifaria US2012/0135477 Bacillus anthracis W02010139719 SEQ IDNo. 8 US2012/0135477 Frankia alni WO2010139719 SEQ ID No. 9US2012/0135477 Rhodopseudomonas WO2010139719  SEQ ID No. 10 palentUS2012/0135477

TABLE 13 WT AacSHC amino acid and nucleotide SEQ ID No. Amino acidNucleotide SEQ ID No. Strain SEQ ID No. SEQ ID No 1 WT AacSHC 169Alicyclobacillus acidocaldarius AB007002.1 SEQ ID No 1 (GI: 218288697)165 Alicyclobacillus acidocaldarius ZP_03492960.1

TABLE 14 AacSHC Derivative arnno acid and nucleotide SEQ ID No. SHCNucleotide Amino acid Derivative SEQ ID SEQ ID No. Mutation (s) name No.SEQ ID No. 5 T77A 6 SEQ ID No. 7 I92V 8 SEQ ID No. 9 F129L 10 SEQ ID No.11 M132R 12 SEQ ID No. 13 A224V 14 SEQ ID No. 15 I432T 16 SEQ ID No. 17Q579H 18 SEQ ID No. 19 F601Y 20 SEQ ID No. 171 F605W 170 SEQ ID No. 21M132R + A224V + I432T 215G2 22 SEQ ID No. 23 M132R + I432T SHC26 24 SEQID No. 25 F601Y SHC3 26 SEQ ID No. 27 T77A + I92V + F129L 111C8 28 SEQID No. 29 Q579H + F601Y 101A10 30 SEQ ID No. 31 F129L SHC10 32 SEQ IDNo. 33 F129L + F601Y SHC30 34 SEQ ID No. 35 F129L + M132R + I432T SHC3136 SEQ ID No. 37 M132R + I432T + F601Y SHC32 38 SEQ ID No. 39 F129L +M132R + I432T + SHC33 40 F601Y

TABLE 15 WT ZmoSHC1 and ZmoSHC1 Derivative Amino acid and nucleotide SEQID No. Amino acid SHC Derivative Nucleotide SEQ ID No.Strain/mutation(s) name SEQ ID No. SEQ ID No. 2 WT ZmoSHC1 ReferenceZmoSHC1 166 ZmoSHC1 (GI: 56552444) sequence ATCC 31821] SEQ ID No. 2 inWO 2010/139719 SEQ ID No. 41 S129A 42 SEQ ID No. 43 V145V 44 SEQ ID No.45 F182L 46 SEQ ID No. 47 Y185R 48 SEQ ID No. 49 G282V 50 SEQ ID No. 511498T 52 SEQ ID No. 53 H646H 54 SEQ ID No. 55 F668Y 56 SEQ ID No. 57Y185R + G282V + I498T   215G2 ZM1 58 SEQ ID No. 59 Y185R + I498T  SHC26ZM1 60 SEQ ID No. 61 F668Y    SHC3 ZM1 62 SEQ ID No. 63 S129A + V145V +F182L   111C8 ZM1 64 SEQ ID No. 65 H646H + F668Y 101A10 ZM1 66 SEQ IDNo. 67 F182L  SHC10 ZM1 68 SEQ ID No. 69 F182L + F668Y  SHC30 ZM1 70 SEQID No. 71 F182L + Y185R + I498T  SHC31 ZM1 72 SEQ ID No. 73 Y185R +I498T + F668Y  SHC32 ZM1 74 SEQ ID No. 75 F182L + Y185R + I498T + F668Y SHC33 ZM1 76 SEQ ID No. 173 F698W 172

TABLE 16 WT ZmoSHC2 and ZmoSHC2 Derivative amino acid and nucleotide SEQID No. Amino acid Nucleotide SEQ in No. Strain/mutation(s) SHCDerivative name SEQ ID No. SEQ ID No. 3 WT ZmoSHC2 Reference ZmoSHC1 167ZmoSHC2 (GI: 677871) sequence SEQ ID No. 77 G85A 78 SEQ ID No. 79 V100V80 SEQ ID No. 81 F137L 82 SEQ ID No. 83 I140R 84 SEQ ID No. 85 V233V 86SEQ ID No. 87 I450T 88 SEQ ID No. 89 N598H 90 SEQ ID No. 91 F620Y 92 SEQID No. 93 I140R + V233V + I450T 215G2ZM2 94 SEQ ID No. 95 I140R +1450TSHC26 ZM2  96 SEQ ID No. 97 F620Y  SHC3 ZM2 98 SEQ ID No. 99 G85A +V100V + F137L  111C8 ZM2 100 SEQ ID No. 101 N598H + F620Y 101A10 ZM2 102 SEQ ID No. 103 F137L SHC10 ZM2 104 SEQ ID No. 105 F137L + F620YSHC30 ZM2 106 SEQ ID No. 107 F137L + I140R + I450T SHC31 ZM2 108 SEQ IDNo. 109 I140R + I450T + F620Y SHC32 ZM2 110 SEQ ID No. 111 F137L +I140R + I450T + F620Y SHC33 ZM2 112 SEQ ID No. 175 F624W 174

TABLE 17 WT BjpSHC1 and BjpSHC1 Derivative amino acid and nucleotide SEQID No. SRC Nucleotide Amino acid Derivative SEQ ID SEQ ID No.Strain/mutation(s) name No. SEQ ID No. 4 WT B. japonicum SHC 168(GI:ABQ33590.1) SEQ ID No. 113 A88A 114 SEQ ID No. 115 V104V 116 SEQ IDNo. 117 F141L 118 SEQ ID No. 119 Y144R 120 SEQ ID No. 121 V241V 122 SEQID No. 123 I459T 124 SEQ ID No. 125 M607H 126 SEQ ID No. 127 F628Y 128SEQ ID No. 129 Y144R + V241V + I459T  215G2 Bjp 130 SEQ ID No. 131Y144R + I459T  SHC26 Bjp 132 SEQ ID No. 133 F628Y  SHC3 Bjp 134 SEQ IDNo. 135 A88A + V104V + F141L  111C8 Bjp 136 SEQ ID No. 137 M607H + F628Y101A10 Bjp  138 SEQ ID No. 139 F141L SHC10 Bjp 140 SEQ ID No. 141F141L + F628Y SHC30 Bjp 142 SEQ ID No. 143 F141L + Y144R + I459T SHC31Bjp 144 SEQ ID No. 145 M144R + I459T + F628Y SHC32 Bjp 146 SEQ ID No.147 F141L + Y144R + I459T + SHC33 Bjp 148 F628Y SEQ ID No. 177 F658W 176

(Alicyclobacillus acidocaldarius), AacSHC SEQ ID No. 1MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR (Zymomonas mobilis), ZmoSHC1SEQ ID No. 2 MGIDRMNSLSRLLMKKIFGAEKTSYKPASDTIIGTDTLKRPNRRPEPTAKVDKTIFKTMGNSLNNTLVSACDWLIGQQKPDGKWVGAVESNASMEAEWCLALWFLGLEDHPLRPRLGNALLEMQREDGSWGVYFGAGNGDINATVEAYAALRSLGYSADNPVLKKAAAWIAEKGGLKNIRVFTRYWLALIGEWPWEKTPNLPPEIIWFPDNFVFSIYNFAQWARATMVPIAILSARRPSRPLRPQDRLDELFPEGRARFDYELPKKEGIDLWSQFFRTTDRGLHWVQSNLLKRNSLREAAIRHVLEWIIRHQDADGGWGGIQPPWVYGLMALHGEGYQLYHPVMAKALSALDDPGWRHDRGESSWIQATNSPVWDTMLALMALKDAKAEDRFTPEMDKAADWLLARQVKVKGDWSIKLPDVEPGGWAFEYANDRYPDTDDTAVALIALSSYRDKEEWQKKGVEDAITRGVNWLIAMQSECGGWGAFDKDNNRSILSKIPFCDFGESIDPPSVDVTAHVLEAFGTLGLSRDMPVIQKAIDYVRSEQEAEGAWFGRWGVNYIYGTGAVLPALAAIGEDMTQPYITKACDWLVAHQQEDGGWGESCSSYMEIDSIGKGPTTPSQTAWALMGLIAANRPEDYEAIAKGCHYLIDRQEQDGSWKEEEFTGTGFPGYGVGQTIKLDDPALSKRLLQGAELSRAFMLRYDFYRQFFPIMALSRAERLIDLNN (Zymomonas mobilis), ZmoSHC2 SEQ ID No. 3MTVSTSSAFHHSPLSDDVEPIIQKATRALLEKQQQDGHWVFELEADATIPAEYILLKHYLGEPEDLEIEAKIGRYLRRIQGEHGGWSLFYGGDLDLSATVKAYFALKMIGDSPDAPHMLRARNEILARGGAMRANVFTRIQLALFGAMSWEHVPQMPVELMLMPEWFPVHINKMAYWARTVLVPLLVLQALKPVARNRRGILVDELFVPDVLPTLQESGDPIWRRFFSALDKVLHKVEPYWPKNMRAKAIHSCVHFVTERLNGEDGLGAIYPAIANSVMMYDALGYPENHPERAIARRAVEKLMVLDGTEDQGDKEVYCQPCLSPIWDTALVAHAMLEVGGDEAEKSAISALSWLKPQQILDVKGDWAWRRPDLRPGGWAFQYRNDYYPDVDDTAVVTMAMDRAAKLSDLHDDFEESKARAMEWTIGMQSDNGGWGAFDANNSYTYLNNIPFADHGALLDPPTVDVSARCVSMMAQAGISITDPKMKAAVDYLLKEQEEDGSWFGRWGVNYIYGTWSALCALNVAALPHDHLAVQKAVAWLKTIQNEDGGWGENCDSYALDYSGYEPMDSTASQTAWALLGLMAVGEANSEAVTKGINWLAQNQDEEGLWKEDYYSGGGFPRVFYLRYHGYSKYFPLWALARYRNLKKAN QPIVHYGM(Bradyrhizobium japonicum), BjpSHC SEQ ID No. 4MTVTSSASARATRDPGNYQTALQSTVRAAADWLIANQKPDGHWVGRAESNACMEAQWCLALWFMGLEDHPLRKRLGQSLLDSQRPDGAWQVYFGAPNGDINATVEAYAALRSLGFRDDEPAVRRAREWIEAKGGLRNIRVFTRYWLALIGEWPWEKTPNIPPEVIWFPLWFPFSIYNFAQWARATLMPIAVLSARRPSRPLPPENRLDALFPHGRKAFDYELPVKAGAGGWDRFFRGADKVLHKLQNLGNRLNLGLFRPAATSRVLEWMIRHQDFDGAWGGIQPPWIYGLMALYAEGYPLNHPVLAKGLDALNDPGWRVDVGDATYIQATNSPVWDTILTLLAFDDAGVLGDYPEAVDKAVDWVLQRQVRVPGDWSMKLPHVKPGGWAFEYANNYYPDTDDTAVALIALAPLRHDPKWKAKGIDEAIQLGVDWLIGMQSQGGGWGAFDKDNNQKILTKIPFCDYGEALDPPSVDVTAHIIEAFGKLGISRNHPSMVQALDYIRREQEPSGPWFGRWGVNYVYGTGAVLPALAAIGEDMTQPYIGRACDWLVAHQQADGGWGESCASYMDVSAVGRGTTTASQTAWALMALLAANRPQDKDAIERGCMWLVERQSAGTWDEPEFTGTGFPGYGVGQTIKLNDPALSQRLMQGPELSRAFMLRYGMYRHYFPLMALGRALRPQSHS (Burkholderia ambifaria)SEQ ID No. 149 MNDLTEMATLSAGTVPAGLDAAVASATDALLAAQNADGHWVYELEADSTIPAEYVLLVHYLGETPNLELEQKIGRYLRRVQQADGGWPLFTDGAPNISASVKAYFALKVIGDDENAEHMQRARRAIQAMGGAEMSNVFTRIQLALYGAIPWRAVPMMPVEIMLLPQWFPFHLSKVSYWARTVIVPLLVLNAKRPIAKNPRGVRIDELFVDPPVNAGLLPRQGHQSPGWFAFFRVVDHALRAADGLFPNYTRERAIRQAVSFVDERLNGEDGLGAIYPAMANAVMMYDVLGYAEDHPNRAIARKSIEKLLVVQEDEAYCQPCLSPVWDTSLAAHALLETGDARAEEAVIRGLEWLRPLQILDVRGDQISRRPHVRPGGWAFQYANPHYPDVDDTAVVAVAMDRVQKLKHNDAFRDSIARAREWVVGMQSSDGGWGAFEPENTQYYLNNIPFSDHGALLDPPTADVSGRCLSMLAQLGETPLNSEPARRALDYMLKEQEPDGSWYGRWGMNYVYGTWTALCALNAAGLTPDDPRVKRGAQWLLSIQNKDGGWGEDGDSYKLNYRGFEQAPSTASQTAWALLGLMAAGEVNNPAVARGVEYLIAEQKEHGLWDETRFTATGFPRVFYLRYHGYRKFFPLWALARYRNLKRNNA TRVTFGL(Burkholderia ambifaria) SEQ ID No. 151MIRRMNKSGPSPWSALDAAIARGRDALMRLQQPDGSWCFELESDATITAEYILMMHFMDKIDDARQEKMARYLRAIQRLDTHGGWDLYVDGDPDVSCSVKAYFALKAAGDSEHAPHMVRARDAILELGGAARSNVFTRILLATFGQVPWRATPFMPIEFVLFPKWVPISMYKVAYWARTTMVPLLVLCSLKARARNPRNIAIPELFVTPPDQERQYFPPARGMRRAFLALDRVVRHVEPLLPKRLRQRAIRHAQAWCAERMNGEDGLGGIFPPIVYSYQMMDVLGYPDDHPLRRDCENALEKLLVTRPDGSMYCQPCLSPVWDTAWSTMALEQARGVAVPEAGAPASALDELDARIARAYDWLAERQVNDLRGDWIENAPADTQPGGWAFQYANPYYPDIDDSAVVTAMLDRRGRTHRNADGSHPYAARVARALDWMRGLQSRNGGFAAFDADCDRLYLNAIPFADHGALLDPPTEDVSGRVLLCFGVTKRADDRASLARAIDYVKRTQQPDGSWWGRWGTNYLYGTWSVLAGLALAGEDPSQPYIARALAWLRARQHADGGWGETNDSYIDPALAGTNAGESTSNCTAWALLAQMAFGDGESESVRRGIAYLQSVQQDDGFWWHRSHNAPGFPRIFYLKYHGYTAYFPLWALARYRRLAGGVSAAGAHAVPASTGADAALA (Bacillus anthracis) SEQ ID No. 153MLLYEKAHEEIVRRATALQTMQWQDGTWRFCFEGAPLTDCHMIFLLKLLGRDKEIEPFVERVASLQTNEGTWKLHEDEVGGNLSATIQSYAALLASKKYTKEDANMKRAENFIQERGGVARAHFMTKFLLAIHGEYEYPSLFHLPTPIMFLQNDSPFSIFELSSSARIHLIPMMLCLNKRFRVGKKLLPNLNHIAGGGGEWFREDRSPVFQTLLSDVKQIISYPLSLHHKGYEEIERFMKERIDENGTLYSYATASFYMIYALLALGHSLQSSMIQKAIAGITSYIWKMERGNHLQNSPSTVWDTALLSYALQEAQVSKDNKMIQNATAYLLKKQHTKKADWSVHAPALTPGGWGFSDVNTTIPDIDDTTAVLRALARSRGNKNIDNAWKKGGNWIKGLQNNDGGWGAFEKGVTSKLLAKLPIENASDMITDPSTPDITGRVLEFFGTYAQNELPEKQIQRAINWLMNVQEENGSWYGKWGICYLYGTWAVMTGLRSLGIPSSNPSLTRAASWLEHIQHEDGGWGESCHSSVEKRFVTLPFSTPSQTAWALDALISYYDTETPAIRKGVSYLLSNPYVNERYPTGTGLPGAFYIRYHSYA HIYPLLTLAHYIKKYRK(Frankia alni) SEQ ID No. 155MPAGVGVLVWLDQRLRAMGRPDLVTTTGGAEIPFVLVAATASTVGVALALRRPRHPVGWLFLALGGVLLLSGGTQGYAAYGAVARPGRLPAADLVAIYADAGFIPWLVLVALILHLTPTGRPLSARWGRIALATAVAGGLWLLVGLVTTETMQPPFQSVTNPLLIGGPLGPLLVARRVLGLATGAGVVLAAVSLIVRFRRSVDVERRQLLWVAVAAVPLPVLMAASFAASYAGNNTAAGLAAATLIGLLAIGAGLAIGQYHLYDVEEILSRAVTYLLVSGLLAASYATVVIVVGQSLAGRTGRSQISAVLATLAAVAVTAPAYRKIQEGVDRRFSRRRFETLQVIRRYLRDPDPDVAVEEVLRRALGDPTLAVAYLVDDRRQWVSADGQPANPGNSFMAAVEVYRRGRPIARVTFDRGRAQPGLVRAAATAATAELDNAGLRAAVALQLVEVRQSRTRIAAAQFAERRTIERNLHDGAQQRLLALALQLRAVQLGGDEASLRQAISTGIDQLQAAVVELRELANGLHPAVLADGGLAAALDDVAARTPVPIKISAPDRRYPPDLEAAAWFIACEAMANAVKHAHPTTIAVDVSAPDGQLIVEVRDDGIGGAQPSGPGLRGIADRAEAFGGSLTVHTDPGTGTTIRALLHRRSPLSSGRRSVMIEGCVDVVAVRRFRCRSSRGSGSRRRRSSWRCGGICGS RCRTGMSRSCSRNAASKLIT(Rhodopseudomonas palent) SEQ ID No. 157MDSILAPRADAPRNIDGALRESVQQAADWLVANQKPDGHWVGRAETNATMEAQWCLALWFLGLEDHPLRVRLGRALLDTQRPDGAWHVFYGAPNGDINATVEAYAALRSLGHRDDEEPLRKARDWILSKGGLANIRVFTRYWLALIGEWPWEKTPNILPEVIWLPTWFPFSIYNFAQWARATLMPIAVLSAHRPSRPLAPQDRLDALFPQGRDSFNYDLPARLGAGVWDVIFRKIDTILHRLQDWGARRGPHGIMRRGAIDHVLQWIIRHQDYDGSWGGIQPPWIYGLMALHTEGYAMTHPVMAKALDALNEPGWRIDIGDATFIQATNSPVWDTMLSLLAFDDAGLGERYPEQVERAVRWVLKRQVLVPGDWSVKLPDVKPGGWAFEYANNFYPDTDDTSVALMALAPFRHDPKWQAEGIEDAIQRGIDWLVAMQCKEGGWGAFDKDNDKKILAKIPFCDFGEALDPPSADVTAHIIEAFAKVGLDRNHPSIVRALDYLKREQEPEGPWFGRWGVNYVYGTGAVLPALAAIGEDMRQPYIARACDWLIARQQANGGWGESCVSYMDAKQAGEGTATASQTAWALMALIAADRPQDRDAIERGCLYLTETQRDGTWQEVHYTGTGFPGYGVGQTIKLNDPLLSKRLMQGPELSRSFMLRYDLYRHYFPMMAIGRVLRQRGDRSGH (Streptomyces coelicolor)SEQ ID No. 159 MTATTDGSTGASLRPLAASASDTDITIPAAAAGVPEAAARATRRATDFLLAKQDAEGWWKGDLETNVTMDAEDLLLRQFLGIQDEETTRAAALFIRGEQREDGTWATFYGGPGELSTTIEAYVALRLAGDSPEAPHMARAAEWIRSRGGIASARVFTRIWLALFGWWKWDDLPELPPELIYFPTWVPLNIYDFGCWARQTIVPLTIVSAKRPVRPAPFPLDELHTDPARPNPPRPLAPVASWDGAFQRIDKALHAYRKVAPRRLRRAAMNSAARWIIERQENDGCWGGIQPPAVYSVIALYLLGYDLEHPVMRAGLESLDRFAVWREDGARMIEACQSPVWDTCLATIALADAGVPEDHPQLVKASDWMLGEQIVRPGDWSVKRPGPPGGWAFEFHNDNYPDIDDTAEVVLALRRVRHHDPERVEKAIGRGVRWNLGMQSKNGAWGAFDVDNTSAFPNRLPFCDFGEVIDPPSADVTAHVVEMLAVEGLAHDPRTRRGIQWLLDAQETDGSWFGRWGVNYVYGTGSVIPALTAAGLPTSHPAIRRAVRWLESVQNEDGGWGEDLRSYRYVREWSGRGASTASQTGWALMALLAAGERDSKAVERGVAWLAATQREDGSWDEPYFTGTGFPWDFSINYNLYRQVFPLTALGRYVHGEPFAKKPRAADAPAEAAPAEVKGS SEQ ID No. 169ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant 101A10(SEQ ID No. 30) ATGGCTGAGCAGTTGGTGGAAGCACCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACGTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCAGGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGGTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCATTACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTACCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant 101A10(SEQ ID No. 29) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVHYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant 111C8 ((SEQ ID No. 28)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCGCGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCGTCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGCTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATCACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant 111C8(SEQ ID No. 27) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGAWALYPGGPPDLDTTVEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRQIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC215G2 (SEQ ID No. 22)ATGGCTGAGCAGTTGGTGGAAGCTCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGAGGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGTGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACACCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC215G2(SEQ ID No. 21) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRVLHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC3 (SEQ ID No. 26)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGAGATGAGGCAGCAGCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTACCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC3(SEQ ID No. 25) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC10 (SEQ ID No. 32)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGCTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC10(SEQ ID No. 31) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC26 (SEQ ID No. 24)ATGGCTGAGCAGTTGGTGGAAGCTCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGAGGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACACCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC26(SEQ ID No. 23) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQIERR Variant SHC30 (SEQ ID No. 34)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGCTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTACCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC30(SEQ ID No. 33) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC31 (SEQ ID No. 36)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGCTCACGCGGAGGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACACCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC31(SEQ ID No. 35) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRRWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC32 (SEQ ID No. 38)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGAGGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACACCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGGAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTACCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC32(SEQ ID No. 37) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC33 (SEQ ID No. 40)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGCTCACGCGGAGGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACATCGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACACCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTACCCAGGGGATTTCTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant SHC33(SEQ ID No. 39) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRRWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant F605W (SEQ ID No. 170)ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAGGACGAAGGCTACTGGTGGGGGCCGCTTCTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGATCGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTACCCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAGCCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGATGTGGCTGGCGCTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAACATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTGCCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGGATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGCGCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATCGCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTGGATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCGGGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGGGCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACGGCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGCTGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATCCCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTCGGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTCGGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAGCCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTACGAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGCAGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCGTACTACACCGGCACGGGCTTCCCAGGGGATTGGTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTCGGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant F605W(SEQ ID No. 171) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR (ZmoSHC1) SEQ ID No. 166ATGGGTATTGACAGAATGAATAGCTTAAGTCGCTTGTTAATGAAGAAGATTTTCGGGGCTGAAAAAACCTCGTATAAACCGGCTTCCGATACCATAATCGGAACGGATACCCTGAAAAGACCGAACCGGCGGCCTGAACCGACGGCAAAAGTCGACAAAACGATATTCAAGACTATGGGGAATAGTCTGAATAATACCCTTGTTTCAGCCTGTGACTGGTTGATCGGACAACAAAAGCCCGATGGTCATTGGGTCGGTGCCGTGGAATCCAATGCTTCGATGGAAGCAGAATGGTGTCTGGCCTTGTGGTTTTTGGGTCTGGAAGATCATCCGCTTCGTCCAAGATTGGGCAATGCTCTTTTGGAAATGCAGCGGGAAGATGGCTCTTGGGGAGTCTATTTCGGCGCTGGAAATGGCGATATCAATGCCACGGTTGAAGCCTATGCGGCCTTGCGGTCTTTGGGGTATTCTGCCGATAATCCTGTTTTGAAAAAAGCGGCAGCATGGATTGCTGAAAAAGGCGGATTAAAAAATATCCGTGTCTTTACCCGTTATTGGCTGGCGTTGATCGGGGAATGGCCTTGGGAAAAGACCCCTAACCTTCCCCCTGAAATTATCTGGTTCCCTGATAATTTTGTCTTTTCGATTTATAATTTTGCCCAATGGGCGCGGGCAACCATGGTGCCGATTGCTATTCTGTCCGCGAGACGACCAAGCCGCCCGCTGCGCCCTCAAGACCGATTGGATGAACTGTTTCCAGAAGGCCGCGCTCGCTTTGATTATGAATTGCCGAAAAAAGAAGGCATCGATCTTTGGTCGCAATTTTTCCGAACCACTGACCGTGGATTACATTGGGTTCAGTCCAATCTGTTAAAGCGCAATAGCTTGCGTGAAGCCGCTATCCGTCATGTTTTGGAATGGATTATCCGGCATCAGGATGCCGATGGCGGTTGGGGTGGAATTCAGCCACCTTGGGTCTATGGTTTGATGGCGTTACATGGTGAAGGCTATCAGCTTTATCATCCGGTGATGGCCAAGGCTTTGTCGGCTTTGGATGATCCCGGTTGGCGACATGACAGAGGCGAGTCTTCTTGGATACAGGCCACCAATAGTCCGGTATGGGATACAATGTTGGCCTTGATGGCGTTAAAAGACGCCAAGGCCGAGGATCGTTTTACGCCGGAAATGGATAAGGCCGCCGATTGGCTTTTGGCTCGACAGGTCAAAGTCAAAGGCGATTGGTCAATCAAACTGCCCGATGTTGAACCCGGTGGATGGGCATTTGAATATGCCAATGATCGCTATCCCGATACCGATGATACCGCCGTCGCTTTGATCGCCCTTTCCTCTTATCGTGATAAGGAGGAGTGGCAAAAGAAAGGCGTTGAGGACGCCATTACCCGTGGGGTTAATTGGTTGATCGCCATGCAAAGCGAATGTGGCGGTTGGGGAGCCTTTGATAAGGATAATAACAGAAGTATCCTTTCCAAAATTCCTTTTTGTGATTTCGGAGAATCTATTGATCCGCCTTCAGTCGATGTAACGGCGCATGTTTTAGAGGCCTTTGGCACCTTGGGACTGTCCCGCGATATGCCGGTCATCCAAAAAGCGATCGACTATGTCCGTTCCGAACAGGAAGCCGAAGGCGCGTGGTTTGGTCGTTGGGGCGTTAATTATATCTATGGCACCGGTGCGGTTCTGCCTGCTTTGGCGGCGATCGGTGAAGATATGACCCAGCCTTACATCACCAAGGCTTGCGATTGGCTGGTCGCACATCAGCAGGAAGACGGCGGTTGGGGCGAAAGCTGCTCTTCCTATATGGAGATTGATTCCATTGGGAAGGGCCCAACCACGCCGTCCCAGACTGCTTGGGCTTTGATGGGGTTGATCGCGGCCAATCGTCCCGAAGATTATGAAGCCATTGCCAAGGGATGCCATTATCTGATTGATCGCCAAGAGCAGGATGGTAGCTGGAAAGAAGAAGAATTCACCGGCACCGGATTCCCCGGTTATGGCGTGGGTCAGACGATCAAGTTGGATGATCCGGCTTTATCGAAACGATTGCTTCAAGGCGCTGAACTGTCACGGGCGTTTATGCTGCGTTATGATTTTTATCGGCAATTCTTCCCGATTATGGCGTTAAGTCGGGCAGAGAGACTGATTGATTTGAATAATTGA

TABLE 18 percent sequence identity calculations using Blast and GAPprogram algorithms for WT AacSHC enzyme relative to the WT SHC enzymesdisclosed in WO2010/0139719 (BASF). Sequence alignment of WT AacSHC % %Source of (631AA, SEQ ID identity identity WT SHC Length No. 1) versusBlast maps GAP (HAC) (AA) WO 2010139719 44% 40% Zymomonas 725 SEQ ID No.1 mobilis WO 2010139719 44% 40% Zymomonas 725 SEQ ID No. 2 mobilis WO2010139719 44% 42% Brodyrhizobium 684 SEQ ID No. 5 japonicum WO2010139719 41% 38% Burkholderia 657 SEQ ID No. 6 ambifaria6 WO2010139719 40% 37% Burkholderia 682 SEQ ID No. 7 ambifaria WO 201013971934% 32% Bacillus anthracis 617 SEQ ID No. 8 WO 2010139719 37% n.d.Frankia alni 720 SEQ ID No. 9 WO 2010139719 45% 42% Rhodvseudomonas 685SEQ ID No. 10 palent WO 2010139719 52% 46% Streptomyces 679 SEQ ID No.11 coelicolor

TABLE 19 percent sequence identity calculations using Blast and Huangand Miller algorithms for WT AacSHC enzyme relative to the WT ZmoSHC1and WT ZraoSHC2 enzymes disclosed in Seitz (2012) % Percent identityCompared identity as disclosed in Seitz SHC sequences Blast maps (2012,thesis) AacSHC/HAC vs 44% 41% ZmoSHC1 AacSHC/HAC vs 39% 37% ZmoSHC2AacSHC/HAC vs 98% ND AacSHC ZmoSHC1 vs 36% 34% ZmoSHC2 AacSHC vs 39% 37%ZmoSHC2 Miriam Seitz thesis is available athttp://elib.unistuttgart.de/handle/11682/1400

TABLE 20 Nomenclature for Homofarnesol Compound Abbreviation Name andStructure E,E-Homofarnesol EEH

E,Z-Homofarnesol EZH

Z,E-Homofarnesol ZEH

Z,Z-Homofarnesol ZZH

TABLE 21 Nomenclature for reaction products Com- De- pound scriptionName and Structure (I) (−)- Ambrox

(II) Macro- cycle

(III) 9b-epi- Ambrox

(IV) Escher et al (1990)

Escher S, Giersch W., Niclass Y, Bernardinello G and Ohloff G (1990).Configuration- odor relationships in 5β-Ambrox. Helv. Chim. Acta 73,1935-1947.

BRIEF DESCRIPTIONS OF THE DRAWINGS

For a better understanding of the present disclosure, reference is madeto the accompanying drawings in which:

FIGS. 1-4 show the sequence alignment of selected AacSHC Derivativesrelative to AacSHC SEQ ID No. 1 In descending order of appearance, theSEQ ID Nos. of FIG. 1 are: SEQ ID No. 1, SEQ ID No. 29. SEQ ID No. 27,SEQ ID No. 21, SEQ ID No. 19, SEQ ID No. 9, SEQ ID No. 23, SEQ ID No.33, SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 39;

FIG. 5 shows a plasmid map;

FIG. 6 shows the relative HAC activities of the wild-type AacSHC andAacSHC Derivatives as set out in Table 24 under standard conditions(pH6.0, 55° C., 0.050% SDS, cells to OD_(650nm) of 10);

FIG. 7a shows the HAC activity profiles of the AacSHC Derivativesrelative to WTAacSHC using homofarnesol quality EEH:EZH 87:13 and at a96% purity of homofarnesol (as determined using NMR);

FIG. 7b shows the relative improvement of the AacSHC Derivativesrelative to WT SHC (4 h (initial velocity) and yield at 22 h) usinghomofarnesol quality EEH:EZH 87:13 and at a 96% purity of homofarnesol(as determined using NMR);

FIG. 8a shows the HAC activity profiles of the AacSHC Derivativesrelative to WTAacSHC using homofarnesol quality EEH:EZH 92:08 and at a100% purity of homofarnesol (as determined using NMR);

FIG. 8b shows the relative improvement of the AacSHC Derivativesrelative to WT SHC (4 h (initial velocity) and yield at 22 h) usingHomofarnesol quality EEH:EZH 92:08 and at a 100% purity of Homofarnesol(as determined using NM R);

FIG. 9a shows the HAC activity profiles of the AacSHC Derivatives as setout in Table 24 relative to WTAacSHC using Homofarnesol quality EEH:EZH66:33 and at a 76% purity of Homofarnesol (as determined by NMR);

FIG. 9b shows the relative improvement of the AacSHC Derivativesrelative to WT SHC (4 h (initial velocity) and yield at 22 h) usingHomofarnesol quality EEH:EZH 66:33 and at a 76% purity of Homofarnesol(as determined by NMR);

FIG. 10 shows the HAC activity results for three SHC Derivatives showingapprox. 10-fold (215G2), 7-fold (SHC26) and 6-fold (SHC32) improvementover the wild-type AacSHC/HAC enzyme;

FIG. 11 shows the observed E,E-homofarnesol conversion to Ambrox by aSHC/HAC derivative (215G2 SHC) and WT AacSHC. At 7 hours of reaction(estimation of initial reaction velocity) conversion with variant 215G2SHC was 13-fold higher than that achieved with wild-type SHC. At 48hours of reaction conversion with the variant was about 8-fold that ofthe wild-type enzyme;

FIG. 12 shows the reaction products produced (Ambrox and product (IV))when EEH is used as a starting material (for bioconversion with WI SHCand/or a SHC/HAC Derivative); and the reaction products produced((−)-Ambrox (1) and products (II), (IV) and (III) (see Table 21) whenEE:EZ is used as a starting material); for ease of reference, compoundsI-IV can be identified as follows:

-   I:    (3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan    (−)-Ambrox-   II:    (7aS,11aS,Z)-5,8,8,11a-tetramethyl-2,3,6,7,7a,8,9,10,11,11a-decahydrobenzo[b]oxonine-   IV:    (3aS,5aS,9aS,9bS)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan-   III:    (3aRS,5aSR,9aSR,9bSR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan    9-epi-Ambrox

FIG. 13 shows a GC-analysis of the reaction products for Ambrox andproducts (II) (IV) and (III) in Table 25;

FIG. 14 shows GC-analysis of the reaction products for Ambrox andproducts (II), (IV) and (III) in Table 25;

FIG. 15 provides comparative data for 215G2SHC variant activity in thewhole cell bioconversion assay in the presence of either Triton X-100 orSDS;

FIG. 16 shows the percent converted EEH for different SDS/cell ratios;

FIG. 17 shows % EEH conversion in the standard bioconversion reaction(as described in Example 7) for three different SDS concentrations;

FIG. 18 shows % EEH conversion in the standard bioconversion reaction(as described in Example 7) for three different pH values;

FIG. 19 shows the location of the mutations identified in SHC/HACvariants 101A10, 111C8 and 215G2 on the SHC Crystal Structure (incolour): red for variant 215G2; purple (wine red) for variant 101A0 andgreen for variant 111C8. For the amino acids identified at as beingresponsible for the increased activity, the side-chains are highlightedin yellow in the co-crystallized substrate analog. Other mutations foridentified variants with no improved activity are marked in blue. It isnoted that blue mutations are spread about half-half (i.e. 50:50) overthe 2 domains of the enzyme, whereas the beneficial AacSHC mutationswhich were identified are located mostly (apart from one) in domain 2.The only exception is the mutation F601Y which is in the vicinity of theactive site;

FIG. 20 shows the following mutations (in black and white): mutationshaving no beneficial effect on SHC/HAC activity are shown in black, theyare spread over the 2 domains of the SHC enzyme. In grey are shown themutations identified in SHC variants (101A10, 111C8 and 215G2) showingimproved SHC/HAC activity, they are located with only one exception indomain 2 of the SHC enzyme. The side chain of the mutations contributingto the improved activity of the variants are highlighted;

FIG. 21 shows the cloning and expression region of plasmid pET-28a(+);The SEQ ID Nos. for the sequences in FIG. 21 are as follows:

pET 28a (nucleotide sequence): SEQ ID No. 179;

pET 28a (amino acid sequence): SEQ ID No. 180;

pET 28b (nucleotide sequence): SEQ ID No. 181;

pET 28b (amino acid sequence): SEQ ID No. 182;

pET 28c (nucleotide sequence): SEQ ID No. 183; and

pET 28c (amino acid sequence): SEQ ID No. 184.

FIG. 22 shows the volumetric productivity of a 1.5× concentrated EEHbioconversion reaction containing 375 g/l of cells, 188 g/l EEH, 2.33%SDS as compared with the volumetric productivity of a regularbioconversion which was run in parallel at 125 g/l EEH, 250 g/l cells,1.55% SDS (Example 7);

FIG. 23 shows a regular bioconversion (125 g/l EEH, 250 g/l cells, 1.55%SDS) which was run as described in Example 7 but replacing the citricacid buffer pH 5.4 by either 0.5% or 0.9% NaCl, all other reactionparameters being unchanged. A bioconversion in citric acid buffer wasrun in parallel as a control;

FIG. 24 shows the evolution of the solid phase extraction of (−)-Ambroxover the toluene washes as % of the (−)-Ambrox quantity initiallypresent in 200 ml whole reaction broth (due to the volume ratiobroth/toluene, % in the first extract goes over 100%); and

FIG. 25 shows the evolution of the solid phase extraction of (−)-Ambroxover the ethanol washes as a percent of the (−)-Ambrox quantityinitially present. After 4 washes (total 640 ml EtOH, i.e. 3.2× theinitial whole reaction broth volume or 8× the volume of the solidphase), about 99% of (−)-Ambrox initially present in the reaction brothwas recovered.

EXAMPLES

For the avoidance of doubt, all reference to WT SHC and SHC variants arereferences to WT AacSHC (SEQ ID No. 1) and variants thereof (eg. aslisted in Tables 23 and/or Table 24).

Example 1

Biocatalyst Production

Methods 1

SHC Plasmid Preparation

The gene encoding Alicyclobacillus acidocaldarius squalene hopenecyclase (AacSHC) was inserted into plasmid pET-28a(+), where it is underthe control of an IPTG inducible T7-promotor for protein production inEscherichia coli (see FIGS. 5 and 21). The plasmid was transformed intoE. coli strain BL21 (DE3) using a standard heat-shock transformationprotocol.

Erlenmeyer Flask Culture.

For protein production were used either complex (LB) or minimal media.M9 is one example of minimal media, which were successfully used.

Media Preparation

The minimal medium chosen as default was prepared as follows for 350 mlculture: to 35 ml citric acid/phosphate stock (133 g/l KH₂PO₄, 40 g/l(NH₄)₂HPO₄, 17 g/l citric acid. H₂O with pH adjusted to 6.3) was added307 ml H₂O, the pH adjusted to 6.8 with 32% NaOH as required. Afterautoclaving 0.850 ml 50% MgSO₄, 0.035 ml trace elements solution(composition in next section) solution, 0.035 ml Thiamin solution and 7ml 20% glucose were added.

SHC Biocatalyst Production (Biocatalyst Production)

Small scale biocatalyst production (wild-type SHC or SHC variants), 350ml culture (medium supplemented with 50 μg/ml kanamycin) were inoculatedfrom a preculture of the E. coli strain BL21(DE3) containing the SHCproduction plasmid. Cells were grown to an optical density ofapproximately 0.5 (OD_(650nm)) at 37° C. with constant agitation (250rpm).

Protein production was then induced by the addition of IPTG to aconcentration of 300 μM followed by incubation for a further 5-6 hourswith constant shaking. The resulting biomass was finally collected bycentrifugation, washed with 50 mM Tris-HCl buffer pH 7.5. The cells werestored as pellets at 4° C. or −20° C. until further use. In general 2.5to 4 grams of cells (wet weight) were obtained from 1 liter of culture,independently of the medium used.

Fermentations were prepared and run in 750 ml InforsHT reactors. To thefermentation vessel was added 168 ml deionized water. The reactionvessel was equipped with all required probes (pO₂, pH, sampling,antifoam), C+N feed and sodium hydroxide bottles and autoclaved. Afterautoclaving is added to the reactor

-   -   20 ml 10× phosphate/citric acid buffer    -   14 ml 50% glucose    -   0.53 ml MgSO₄ solution    -   2 ml (NH₄)₂SO₄ solution    -   0.020 ml trace elements solution    -   0.400 ml thiamine solution    -   0.200 ml kanamycin stock

The running parameters were set are as follows: pH=6.95, pO₂=40%, T=30°C., Stirring at 300 rpm. Cascade: rpm setpoint at 300, min 300, max1000, flow l/min set point 0.1, min 0, max 0.6. Antifoam control: 1:9.

The fermenter was inoculated from a seed culture to an OD_(650nm) of0.4-0.5. This seed culture was grown in LB medium (+Kanamycin) at 37°C., 220 rpm for 8 h. The fermentation was run first in batch mode for11.5 h, where after was started the C+N feed with a feed solution(sterilized glucose solution (143 ml H₂O+35 g glucose) to which had beenadded after sterilization: 17.5 ml (NH₄)₂SO₄ solution, 1.8 ml MgSO₄solution, 0.018 ml trace elements solution, 0.360 ml Thiamine solution,0.180 ml kanamycin stock. The feed was run at a constant flow rate ofapprox. 4.2 ml. Glucose and NH₄ ⁺ measurements were done externally toevaluate availability of the C- and N-sources in the culture. Usuallyglucose levels stay very low.

Cultures were grown for a total of approx. 25 hours, where they reachedtypically and OD_(650nm) of 40-45. SHC production was then started byadding IPTG to a concentration of approx. 1 mM in the fermenter (as IPTGpulse or over a period of 3-4 hours using an infusion syringe), settingthe temperature to 40° C. and pO₂ to 20%. Induction of SHC productionlasted for 16 h at 40° C. At the end of induction the cells werecollected by centrifugation, washed with 0.1 M citric acid/sodiumcitrate buffer pH 5.4 and stored as pellets at 4° C. or −20° C. untilfurther use.

Results 1a

In general, with all other conditions unchanged the specific activity ofthe produced biocatalyst was higher when a minimal medium was usedcompared with a complex medium. Induction was carried out successfullyat 30 or 37° C. It was noted that when the induction was done at 40-43°C., a biocatalyst of higher specific activity was obtained.

Results 1b

The following Table 22 shows for 2 examples the culture volume, opticaldensity and amount of cells both at induction start and induction end aswell as the amount of biomass collected (wet weight).

TABLE 22 Volume at cells Volume at cells induction start OD_(650 nm)calculated Induction end OD_(650 nm) collected (ml) Induction start (g)(ml) Induction end (g) Example 1 273 40 10.9 342 55 28 Example 2 272 4412.0 341 57 23 OD_(650 nm) at inoculation: 0.45 (Example 1) and 0.40(Example 2). Starting volumes: 205 ml.

Example 2

Preparation of SHC Variants and Activity Screening

Methods 2

For the avoidance of doubt, EE corresponds to (3E,7E); EZ mixturecorresponds to (3Z,7E); ZE corresponds to (7Z,3E); ZZ corresponds to(7Z,3Z); and EEH corresponds to (3E,7E).

An enzyme evolution program was carried out using the wild-type (WT)Alicyclobacillus acidocaldarius SHC (AacSHC) gene as a template (GenBankM73834, Swissprot P33247). A library of about 10500 SHC variants wasproduced and screened for variants showing increased EEH cyclizationability. Screening was run in reactions in citric acid buffer pH 6.0(0.150 ml) containing 4 g/l EEH and 0.050% SDS, at 55° C. and underconstant agitation.

With hits selected for validation a standard test was run in citric acidbuffer pH 6.0 containing 4 g/l EEH 0.050% SDS, cells that had expressedthe SHC variants to an OD_(650nm) of 10.0 in. The final volume was 1 ml,reactions were incubated at 55° C. and vigorously stirred on a magneticstirrer. Reaction sampling over time allowed investigating activityprofiles (EEH conversion to (−)-Ambrox) and as determined by gaschromatography analysis (see analytic methods below).

From this validation round, 3 variants with improved EEH cyclizationactivity (101A10, 111C8 and 215G2) were obtained and then a total of 8mutations were identified on these 3 variants. A mutations study wasthen run to identify which of these mutations were beneficial withregard to EEH cyclization to Ambrox. In addition to this AacSHCderivative, another AacSHC variant was constructed, which contained allof the identified beneficial mutations (SHC33 as outlined in Table 23below). The screening conditions were: 4 g/l EEH cells to an OD_(650nm)of 10.0, SDS to 0.05% and 0.1% (2 concentrations) and the reactions wererun at 55° C. under constant agitation.

Results 2a

TABLE 23 Mutations in evaluated AacSHC Derivative enzymes SHC T77A I92VF129L M132R A224V I432T Q579H F601Y 101A10 X X 111C8 X X X 215G2 X X XSHC3 X SHC10 X SHC26 X X SHC30 X X SHC31 X X X SHC32 X X X SHC33 X X X X

Results 2b

Of the three selected mutations (101A10, 111C8 and 215G2), 215G2 showedthe best activity.

Example 3

Optimized Reaction Conditions with SHC Variants

Reaction Parameters Investigated: Temperature, SDS Concentration and pH

Methods 3

The reaction conditions for the SHC variants derivatives identified inTable 23 were individually optimized with regard to temperature, pH andSDS concentration. To this end, the E. coli cells were transformed withthe plasmid for the production of the individual variants which werecultivated in Erlenmeyer flasks and SHC production induced as describedabove. In this way it was ensured that all cultures contained same orvery similar SHC quantities. Cells were collected by centrifugation,washed with 0.1M citric acid buffer (pH 6.0) and stored at −20° C. untilfurther used.

Results 3

The result of this optimization study is summarized in the below table.An optimization round was also carried out with wild-type SHC.

The following Table 24 shows optimal reaction conditions for thewild-type and each of the variants considered for the characterizationof each SHC/HAC Derivative enzyme.

TABLE 24 Optimal reaction conditions for SHC Derivative enzymesTemperature SHC (° C.) pH [SDS] (weight/weight %) WT 55 (45-60) 6.0(5.6-6.2)  0.030 (0.010-0.075) 101A10 40 (36-50) 6.4 (5.4-7.0)  0.050(0.010-0.10) 111C8 40 (36-50) 6.0 (5.6-6.6)  0.070 (0.010-0.090) 215G235 (32-50) 5.4 (5.0-6.2)  0.060 (0.010-0.10) SHC3 37 (34-50) 5.8(5.4-6.4)  0.020 (0.010-0.060) SHC10 42 (34-55) 6.0 (5.4-6.4)  0.060(0.030-0.10) SHC26 32 (30-50) 5.4 (5.4-6.2)  0.060 (0.020-0.10) SHC30 35(34-50) 6.2 (5.4-7.0) 0.0050 (0.0025-0.070) SHC31 35 (30-50) 5.6(5.4-6.4)  0.050 (0.010-0.10) SHC32 35 (34-50) 5.6 (5.4-6.4)  0.050(0.010-0.10) SHC33 35 (32-50) 5.2 (4.8-6.4)  0.030 (0.0050-0.10)

Discussion 3

Example 3 shows the differences noted in reaction conditions for the SHCDerivatives compared to WT SHC. Significant deviation from the wild-typeSHC for optimal temperature, pH and SDS concentration were observed withthe SHC variants. Only a small number of mutations have a significanteffect on the optimal bioconversion reaction conditions. For thedetermination of individual reaction conditions with the selected SHCvariants, reactions were run at a substrate loading of 4 g/l of EEH andcells that had produced the wild-type or SHC derivatives at an opticaldensity OD_(650nm) of 10.0.

Temperature

The data in Table 24 demonstrate the surprising finding that whilst theWT SHC enzyme has optimal activity at 55° C. (in the range of 45-60°C.), a number of the SHC Derivatives have optimal activity at 35° C.(34-50° C.). The application of the SHC Derivatives of the presentdisclosure in methods for preparing (−)-Ambrox from E,E-homofarnesol atlower reaction temperatures has significant cost advantages for theproduction of (−)-Ambrox at an industrial scale.

Solubilizing Agent

SDS was selected and identified from a long list of possiblesolubilising agents which were not useful in the bioconversion reaction(see Example 14 for more information) SDS is better than eg. TritonX-100 in terms of reaction velocity and yield (both in the test at 4 g/lof EEH and when 125 g/l EEH is used as provided in Example 7).

Example 4

SHC Variant Activity Testing in Comparison to the WT SHC Enzyme UnderStandard Conditions

Methods 4

For comparing the relative activity of the biocatalysts, the productionof the variants (as set out in Table 24) is described as follows. The E.coli cells were transformed with a plasmid for the production of one ofthe SHC variants and the E. coli cells were then cultivated in LB mediumat 37° C. and 280 rpm, grown to an OD_(650nm) of 0.50 and enzymeproduction induced by the addition of IPTG. Induction lasted for 5.5hours at 37° C., 280 rpm. Cells were collected by centrifugation, washedwith 0.1M citric acid pH 6.0 and stored at −20° C. until further use.When comparing the SHC variant activities (see FIG. 6), a sample of thereaction mixture was loaded onto an SDS-PAGE gel for analyzing the SHCcontent of the reactions. This analysis confirmed that all reactionscontained identical amounts of SHC enzyme.

Results 4a

FIG. 6 shows the relative activities of the wild-type and SHC variantsunder standard conditions (pH6.0, 55° C., 0.050% SDS, cells toOD_(650nm) of 10). It was also noted that wild-type SHC and at least thetested SHC variants according to the Examples of the present disclosureare solvent tolerant. This means that selected water non-misciblesolvents (up to almost 100%) may be added to the bioconversion reaction.

Results 4b

Using the 215G2 SHC variant, no notable effect on the activity of thisvariant was observed when NaCl is added to the reaction (concentrationstested 5 to 100 mM (only)). In addition, NaCl addition up to 100 mM orup to 154 mM (0.9% NaCl), showed no negative effect on SHC activity invariant 215G2. These finding suggest that if the bioconversion reactionis carried out in a physiological solution of NaCl (0.9%) or the likeand the pH is maintained at an appropriate value (eg. about 5.4(5.2-5.6)), then the bioconversion reaction may be carried out in theabsence of a buffer but in the presence of a physiological NaCl solutionor the like.

Discussion 4

FIG. 6 illustrates the ranking of the activity of the selected variantsand wild-type SHC enzymes in terms of EEH conversion to (−)-Ambrox.

Example 5

WT SHC and SHC Derivative Activity Profiles

Methods 5

The activity test was run in 0.1 M citric acid buffer in 5 ml volumeunder constant shaking at 900 rpm on a Heidolph Synthesis 1 apparatus.The pH of the buffer used, the temperature at which the reaction was runand the concentration of SDS (sodium dodecyl sulfate) in the reactionwas depending on the SHC variant which was used (wild-type or variant).The optimal conditions for each of the variants tested are summarized inTable 24 above.

A Homofarnesol starting material of 96% purity and a homofarnesolsubstrate with an EEH:EZH ratio of 87:13 was used.

For the avoidance of doubt, an EE:EZ mixture is a mixture of ((3E,7E)and (3Z,7E) isomers.

Results 5

Homofarnesol Used: EEH:EZH 87:13, Purity (NMR): 96%.

The results of the standard test run under optimized conditions areshown in FIG. 7B (activity profiles of the SHC derivatives relative toWT SHC) and FIG. 7A which shows the relative activity improvement of theSHC Derivatives relative to WT SHC (4 h (initial velocity) and yield at22 h).

Homofarnesol Used: EEH:EZH 92:08, Purity (NMR): 100%

The result of the standard test run under optimized conditions was areshown in FIG. 8B (activity profiles of the AacSHC derivatives relativeto WT AacSHC) and FIG. 8A which shows the relative improvement of theAacSHC Derivatives relative to WT SHC (4 h (initial velocity) and yieldat 22 h).

Homofarnesol Used: EEH:EZH 66:33, Purity (NMR): 76%

The result of the standard test run under optimized conditions was areshown in FIG. 9B (activity profiles of the AacSHC derivatives as set outin Table 24 relative to WTAacSHC) and FIG. 9A which shows the relativeimprovement of the AacSHC Derivatives relative to WT SHC (4 h (initialvelocity) and yield at 22 h).

Discussion 5

The main conclusion was that independently of the quality of theHomofarnesol substrate used, the four best SHC derivative enzymes wereranked in the following order: 215GSHC, SHC26, SHC32 and SHC3.

Example 6

Determining the Mass Balance from Reactions Entirely Extracted withSolvent

Method 6

All conditions being unchanged, for each variant 2 reactions were run.Homofarnesol was used as a substrate. After 4 hours and 22 hours ofincubation the reaction product and unreacted substrate was extractedtotally for each of the variants with a total of 6 washes with an equalvolume of tert-Butyl-Methyl Ether (MTBE/tBME). The Homofarnesol andAmbrox content of each of the washes was determined by GC-analysis. Thetotal amount of Ambrox formed and Homofarnesol remaining were calculatedfrom calibration curves that had been prepared using solutions made fromauthentic Ambrox and homofarnesol.

Result 6

The results in FIG. 10 showed that with the substrate used, the 3 bestvariants were confirmed showing approx. 10-fold (215G2), 7-fold (SHC26)and 6-fold (SHC32) improvement over the wild-type SHC enzyme wasobserved.

Example 7

Performance in Biotransformation at 125 g/l E,E-Homofarnesol (EEH)

Method 7

Using 215G2SHC variant, the objective of increasing volumetricproductivity was addressed. A design of a series of experimental (DOE)investigations was run to optimize test reaction conditions includingparameters pH, cell concentration and SDS concentration. The reactionconditions were: 125 g/l of EEH (from Homofarnesol of EE:EZ 86:14), 250g/l of cells, 1.55% SDS, the reaction being run at 35° C. in 0.1 Mcitric acid buffer pH 5.4.

A typical reaction (150 g total volume) is set up as follows: in 0.75liter Infors fermenters. The reaction vessel is loaded with anappropriate amount of Homofarnesol corresponding to 18.75 g EEH, 2.33 gSDS is added from a 15.5% (w/w) solution prepared in 0.1M citric acidbuffer pH 5.4. A cell suspension is prepared from E. coli cells that hadproduced the 215G2 SHC variant by suspending the cells in 0.1M citricacid buffer pH 5.4. After determination of the cell wet weight of thissuspension by centrifugation for 10 min at 10° C. and 17210 g, theappropriate volume of cells is added to the reaction vessel in order tointroduce 37.5 g of cells into the reaction. The volume of the reactionis completed to 150 g with the required amount of reaction buffer. Thereaction is run at 37° C. under constant stirring at 900 rpm. pHregulation is done using 40% citric acid in water. The reaction issampled over time (1 ml), extracted with 5 volumes of MTBE/tBME (5 ml).The homofarnesol and Ambrox content of the reaction was determined by GCanalysis after clarification of the solvent phase by centrifugation(table top centrifuge, 13000 rpm, 2 min), 10-fold dilution intoMTBE/tBME.

The same reaction was carried out with E. coli cells that had producedthe wild-type SHC enzyme. In that case was the reaction run at 55° C. in0.1M citric acid buffer pH 6.0. A summary of the reaction conditions forthis Example is provided in row 2 of Table 24a below. The reactionconditions presented in row 1 of Table 24a below are taken from previousExamples (eg Examples 3-5).

TABLE 24a Row 2 shows the reaction conditions for Example 7 BiocatalystTemperature [SDS] [EEH] (g/l) (cell SHC (° C.) pH (%, w/w) (g/l) wetweight) 215G2 35° C. 5.4 (5.0-6.2) 0.06 4 1.45 cells 215G2 35° C. 5.4(5.0-6.2) 1.55 125  250 cells

Results 7

FIG. 11 shows the observed EEH conversion to Ambrox by the 2 enzymes. At7 hours of reaction (estimation of initial reaction velocity) conversionwith variant 215G2 SHC was 13-fold higher than that achieved withwild-type SHC. At 48 hours of reaction conversion with the variant wasabout 8-fold that of the wild-type enzyme.

General Comments 7

Cell Concentrations

All concentrations of cells (g/l) in the reactions described thisExample are indicated in wet weight of cells. The concentration as cellwet weight (g/l) of a cell suspension is determined after centrifuging asample of this cell suspension for 10 min at 17210 g and 4° C.

Correlation Between g/l Cells and OD_(650nm)

Using 125 g/l EEH bioconversion with the 215G2 SHC or WT SHC the 250 g/lof cells in this reaction correspond to an OD_(650nm) of about 172 inthat reaction. Variations in the ratio of OD_(650nm) to biocatalystamount were observed when different biocatalyst preparations weretested. When the biocatalyst was used in the standard test at 4 g/l EEHbut applying the cells to an OD_(650nm) of 10.0 it was estimated thatOD_(650nm) of 10.0 is equivalent to 1.45 g/l of cells

Discussion 7

The data demonstrates that an optimized and efficient HAC bioconversionprocess has been developed using relatively high EEH substrateconcentrations (125 g/l) compared with the disclosures in the art whereonly a homofarnesol substrate concentration of from around 0.2 g/l (seeJP2009060799) to about 2.36 g/l (10 mM) has been disclosed in the art(see WO2010/139719A2, US2012/0135477A1) and Seitz et al (2012) as citedabove).

Example 8

GC Analytics

Methods 8

Samples were extracted with an appropriate volume of tert-butylmethylether (MBTE/tBME) for quantification of their content in EEH and Ambrox.The solvent fraction was separated from the water phase bycentrifugation prior to analysis with gas chromatography. 1 μl of thesolvent phase was injected (split ratio 3) onto a 30 m× 0.32 mm×0.25 mZebron ZB-5 column. The column was developed at constant flow (4 ml/minH2) with the temperature gradient: 100° C., 15° C./min to 200° C., 120°C./min to 240° C., 4 min at 240° C., which resulted in separation ofAmbrox, EEH and EZH. Inlet temperature was 200° C., detectortemperature: 300° C.

EEH conversion was calculated from the areas of the peaks correspondingto Ambrox and EEH with the following formula:conversion(%)==100×(Area_(Ambrox_Peak)/(Area_(Ambrox_Peak)+Area_(E,E-Homofarensol Peak)))

The identity of the reaction product Ambrox was confirmed by GC-MS(recorded values and intensities: m/z 221 (100%), m/z 97 (40%), m/z 137(3.3%), m/z 43 (2.6%), m/z 41 (2.5%), m/z 55 (2.4%), m/z 95 (1.9%), m/z67 (1.8%), m/z 81 (138%), m/z 222 (1.7%)).

Discussion 8

Product recovery was carried out by either solvent extraction or steamextraction. Solvents used were eg. MTBE or Hexane:Isopropanol (3:2). Thereaction was extracted repeatedly with equal volumes of solvent and thesolvent fractions GC-analyzed until no substrate or product was detectedanymore. In general 5 to 6 washes were sufficient. Alternativelyextraction of reaction products was done by steam extraction.

Example 9

One Pot Reaction

Methods 9

A 200 ml fermentation was run with E. coli BL21 (DE3) transformed withthe pET28a(+) 215G2 SHC plasmid for the production of 215G2 SHC withN-ter HisTag using the standard growth and induction protocol describedabove. At the end of the induction phase, the aeration was switched off,the temperature set to 35° C., the pH to 5.5 with citric acid andstirrer speed to 500 rpm. The volume of the culture was estimated fromall additions made during culture growth (feed and base consumption).According to this volume and to the OD of the culture, an appropriateamount of SDS was added to the fermenter. EEH was added to 4 g/l. Thereaction was sampled over time, the samples (150-300 μl) extracted with700 μl MTBE for GC analysis. EEH was converted to Ambrox directly in theculture broth. The reaction was run for a total of 22.5 days, duringwhich EEH was added repeatedly.

Results 9

When completion was reached, 10.6 g of EEH had been cyclized to Ambrox.The reaction products (structures provided below) was extracted by steamextraction and was recovered quantitatively from the reaction mixture.

Note on Reaction Products

When Homofarnesol EE:EZ 87:13 is converted by SHC, the reaction productsAmbrox, (II), (IV) and (III) as set out in FIG. 12 are produced andreflect the EE:EZ ratio of the starting material.

When EEH is used as a starting material, only (−)-Ambrox (I) and product(IV) are generated.

When EZH (3Z,7E) is used as a starting material, only products (II) and(III) are generated.

However, when a mixture of EEH and EZH are used, Ambrox (I) and products(II), (IV) and (III) are generated.

If a 100% conversion of EE:EZ 66:34 takes place, this will provide66%:34% ((Ambrox+(IV)):((II)+(III)).

When a steam extraction is carried out, it extracts all 4products—Ambrox and products (II), (IV) and (I11) and a crystallizationstep generates Ambrox with 99% purity (GC) in at least a 70% yield.

Discussion 9

The data demonstrate that the production of (−)-Ambrox is possible in abioconversion reaction or a “one pot” reaction system and that aselective enrichment of Ambrox is achieved after steam extraction andcrystallization.

If the homofarnesol starting material is a mixture of EE and EZ (eg.86:14) isomers, then 2 products originate from each of these isomers (4in total) with (−)-Ambrox being by far the main constituent in the crudeproduct, and the dominant constituent in the crystallized material(purity 99.1%). (+)-Ambrox was not detected.

Example 10

Conversion of EE:EZ Homofarnesol Mixtures

For the avoidance of doubt,

EE corresponds to (3E,7E); EZ mixture corresponds to (3Z,7E): ZEcorresponds to (7Z,3E); ZZ corresponds to (7Z,3Z): EEH corresponds to(3E,7E); and EZH corresponds to (3Z,7E).

Methods 10

EE:EZ mixtures were bioconverted under the following reactionconditions: 146 g/l total homofarnesol with 250 g/l cells and 1.55% SDSusing the following homofarnesol substrates (EE:EZ homofarnesolmixtures):

EE:EZ 86:14 (highest EEH content for this Example),

EE:EZ 69:31 (lowest EEH content for this Example).

EE:EZ 80:20

EEH:EZH 70:30

Bioconversion of 7E, 3E/7E, 3Z Homofarnesol Mixture

Bioconversion was undertaken using the following reaction conditions:

The reaction (150.1 g total volume) was run in 0.1 M citric acid/sodiumcitrate buffer pH 5.4 in an InforsHT 750 ml fermenter contained 146 g/ltotal homofarnesol using a homofarnesol substrate, which was a mixtureof 7E,3E:7E,3Z of 86:14, 250 g/l cells (produced in accordance with themethod of Example 1) and 1.55% SDS. The reaction was run at 35° C. withconstant stirring (800 rpm), pH control was done using 10 to 40% citricacid in water. The reaction mixture was sampled over time, the samplesolvent extracted for GC analysis. It was noted that Homofarnesolconversion went equally fast with the 2 qualities of Homofarnesol (EE:EZ86:14 and EE:EZ 69:31)

Results 10

The conversion of both E,E- and E,Z-Homofarnesol was observed when abioconversion of 125 g/l E,E-Homofarnesol from the EEH:EZH 86:14material using the WT SHC and one specific SHC Derivative (21502 SHC)was carried out. That is, the wild-type SHC enzyme from Alicyclobacillusacidocaldarius produces the same reaction products (i.e. Ambrox,products (II), (IV) and (III)) from EEH:EZH 86:14 material as does anSHC variants from Table 23 from EEH:EZH mixtures. FIGS. 13 and 14provide a GC-analysis of the reaction products for Ambrox and products(II), (IV) and (III).

Discussion 10

The bioconversion of homofarnesol to Ambrox according to the presentdisclosure produces (−)-Ambrox as a predominant compound but may alsoproduce compounds other than (−)-Ambrox (eg. compounds (II) (IV) and(III)) as identified above which may or may not impart pleasantolfactive notes to the (−)-Ambrox product. As demonstrated above, underselective crystallization conditions, Ambrox is separable from other byproducts ((II), (IV) and (III)). Accordingly, if products contribute ina negative matter to the sensory character of the Ambrox end product,the selective separation of products (II), (IV) and (III) from the(−)-Ambrox end product increase its value as a fragrance or flavor orcosmetic or consumer care product. Sensory analysis is carried out usingwell established sensory tests utilized by trained Perfumers. The purityof the (−)-Ambrox end product may be an indicator of the olfactivequality of the product if the product on its own is mainly responsiblefor the desired sensory profile

Example 11

EEH Conversion from a EE:EZ:ZE:ZZ-Homofarnesol Mixture

Methods 11

EE:EZ:ZE:ZZ-Homofarnesol 40:26:20:14 was used as a substrate for EEHconversion with 215G2 SHC. For comparative purposes, other Homofarnesolof EE:EZ 2:1 or 93:07 were also used.

The conversion of the EE:EZ:ZE:ZZ-Homofarnesol mixture was investigatedwith the 215G2 SHC variant but not under optimized conditions. Thereaction conditions were pH 5.8 in 100 mM citrate buffer, 0.10% SDS, 40°C. The following EEH conversions were observed, all reactions being runwith constant 2 g/l EEH (accordingly variable total Homofarnesolconcentrations)

Results 11

The following homofarnesol isomer mixture conversion rates wereobserved:

EE:EZ 2:1 50-55% EE:EZ 93:7    78% EE:EZ:ZE:ZZ 40:26:20:14   6%

Discussion 11

Beyond the yields observed, the data demonstrates that the 215G2 SHCvariant is capable of converting EEH to Ambrox from a complexEE:EZ:ZE:ZZ Homofarnesol mixture. As expected, a lower conversion rateresulting in a lower Ambrox yield was observed. This result isconsistent with the view that homofarnesol isomers other than EEH maycompete with EEH for access to the SHC/HAC derivative enzyme and maythus act as competitive inhibitors and/or alternative substrates for theconversion of EEH to (−)-Ambrox.

Example 12

Comparative Data for Whole Cell Bioconversions Using Triton X-100 andSDS

Methods 12

E. coli host cells were grown according to the protocol in Methods 4 ofExample 4. The bioconversion reaction using the 215G2SHC variant wascarried out according to the standard test in Example 4. Thehomofarnesol substrate at 4 g/l with cells to OD_(650nm) of 10.0 incitric acid/sodium phosphate buffer 0.1M pH5.4, 35° C. and SDS at 0.07%were chosen as the most suitable reaction conditions for the 215G2 SHCvariant.

Results 12

FIG. 15 provides a comparison of the activity of the 215G2SHC variant inthe whole cell bioconversion assay when using Triton X-100 at aconcentration range of 0.005% to 0.48% and SDS at a concentration of0.07%.

Discussion 12

The data demonstrates that maximal activity with Triton X-100 was onlyaround 20% of the activity obtained with SDS.

Example 13

SDS/Cells Ratio

Methods 13

The bioconversion reaction was set up according to Methods 4 in Example4 using EEH substrate at 4 g/i, cells at an OD_(650nm) of 50 that hadproduced the 215G2 SHC derivative enzyme.

Results 13

The results are set out in FIG. 16 which shows the percent converted EEHfor different SDS/cells ratios.

FIG. 16 demonstrates that the percent EEH conversion to (−)-Ambrox usingdifferent SDS/cells ratio values is dependent on the SDS/cells ratio.This ratio has to be carefully set to achieve maximum conversion.

If, for example, the SDS concentration is too low, a suboptimalhomofarnesol conversion may be observed. On the other hand, if, forexample, the SDS concentration is too high, then there may be a riskthat the biocatalyst is affected through either the disruption of theintact microbial cell and/or a denaturation/inactivation of the SHC/HACenzyme. When the bioconversion reaction was carried out according toMethods 7 in Example 7 using 125 g/l EEH and 250 g/l biocatalyst, thebest bioconversion protocol shows a [SDS]/[cells] ratio of 16:1.

Discussion 13

The results demonstrate that there is a degree of interdependencybetween the solubilising agent (SDS) concentration, the biomass amountand the substrate (EEH) concentration. By way of example, as theconcentration of homofarnesol substrate increases, sufficient amounts ofbiocatalyst and solubilising agent (SDS) are required for an efficientbioconversion reaction to take place.

Example 14

Testing of Possible Solubilizing Agents for Use in the BioconversionReaction

Methods 14

Various solubilizing agents (as outlined in Table 26 below) were testedin 215G2 SHC EEH cyclization reactions using the same conditions as inthe standard test (4 g/l EEH, cells to an OD_(650nm) of 10.0) as apossible substitute for SDS. The possibility of enhancing activity(cumulative effect) by combining SDS at its optimal concentration(0.060-0.070%) with other solubilising agents used (at the concentrationdetermined individually as optimal from the screening done with thesecompounds (see Table 26 below)) was also tested using the standard test.In addition, some “Deep eutectic solvents” and ionic liquids, which areknown to help in solubilizing water-insoluble compounds were alsotested.

Results 14

The following Table 26 summarizes which solubilizing agents (eg.surfactants, detergents, solubility enhancers and the like) were testedso far in 215G2 SHC EEH cyclization reactions. In no case was animproved activity compared to the control reaction carried out using SDSat a concentration in the range of 0.060-0.070%. Activities observedwith these compounds used alone at the concentration defined as optimalwere only about 20% of what was obtained in control reactions with SDS.It was noted that when no solubilizing agent at all was added 20% EEHconversion was achieved. When SDS was used and an additionalsolubilizing agent was added (at a concentration defined as optimal inthe test), no synergistic effect was observed. Rather, a decrease inpercent EEH conversion was observed. It can be concluded from the studythat under tested conditions the compounds do not improve EEH conversionat all; rather adverse effects on cyclization are obtained, and that SDSis the most useful of the solubilizing agents studied. In addition, nopositive results were obtained from the tests using “deep eutecticsolvents” and ionic liquids, which are known to help in solubilizingwater-insoluble compounds.

TABLE 26 provides a list of solubilizing agents which were tested in thebioconversion reaction Solubilizing agent Concentration range testedCaprylyl sulfobetaine    0.19-3.0% CHAPS    0.020-0.18% Cremophor EL  0.0063-0.5% Dimethyl sulfoxide  0.00032-0.2% Hexadecylpyridinium  0.013-0.5% chloride monohydrate Myristyl sulfobetaine   0.0009-0.03%Nonidet P40    0.005-0.16% Octyl-β-D-glucopyranoside   0.0008-0.6%Palmitylsulfobetaine 0.00000003-0.03% Pluronic P-105   0.000074-0.018%Quaternary ammonium salts      20-160 mM (eg. tetramethyl ammoniumbromide) Sodium taurodeoxycholate hydrate    0.05-0.4% Stepan ®   0.01-0.6% Thesit ®    0.05-0.8% Triton X-100    0.005-0.32% Tween 20   0.05-0.8% Tween 80    0.01-0.4%

Discussion 14

The Applicant selected and identified SDS as a useful solubilising agentfrom a long list of other solubilizing agents which were shown not to beuseful in the homofarnesol to (−)-Ambrox bioconversion reaction of thepresent disclosure.

Example 15

Sensitivity to SDS Concentration in the Bioconversion Reaction

Methods 15

The conditions applied are the conditions of the standard bioconversion(as described in Example 7) at 125 g/l with 250 g/l biocatalyst and1.55% SDS. Two other SDS concentrations (1.40% and 1.70% SDS were alsotested). All SDS concentrations are in weight/weight %.

The standard bioconversion reaction conditions (as described in Example7) at 125 g/l with 250 g/l biocatalyst and 1.55% SDS were also used totest different pH values.

The control was run in pH 5.4 in 0.1 M citric acid buffer. The reactionsrun at lower pH were run with 0.1M acetic acid buffer.

Results 15

The data in FIG. 17 demonstrate that the bioconversion reaction appearsto be less sensitive to changes in SDS concentrations than when the HACactivity was tested in the standard test at 4 g/l EEH and cells appliedto an OD_(650nm) of 10.0.

The data in FIG. 18 demonstrate that when the bioconversion reactionsare applied, the system appears to be less sensitive to pH variationsthan when the HAC activity is tested in the standard test at 4 g/l EEHand cells applied to an OD_(650nm) of 10.0.

Discussion 15

The data demonstrates the robustness of the bioconversion reaction at125 g/l EEH and 250 g/l of cells with regard to the SDS concentrationrange and the pH range tested.

Example 16

Location of the Identified SHC/HAC Mutations on the Crystal Structure

The positions of the mutations identified in the AacSHC/HAC variants aremarked in FIG. 19 as follows: red for variant 215G2; purple (wine red)for variant 101A10 and green for variant 111C8. For the amino acidsidentified at as being responsible for the increased activity, theside-chains are highlighted in yellow in the co-crystallized substrateanalog. Other mutations for identified variants with no improvedactivity are marked in blue. It is noted that blue mutations are spreadabout half-half (i.e. 50:50) over the 2 domains of the enzyme, whereasthe beneficial AacSHC mutations which were identified are located mostly(apart from one) in domain 2. The only exception is the mutation F601Ywhich is in the vicinity of the active site. If only both of the SHC/HACderivative enzymes 215G2 and 111C8 are considered, then all of themutants are located in domain 2. FIG. 20 provides the same informationin black and white.

Results 16

All of the beneficial mutants (red/green/purple) corresponding to2150G2, 111C8, and 101A10 are located mostly (apart from one mutantF601Y) in domain 2 (Wendt et al (1997) Science 277: 1811) of the SHCcrystal structure (as provided in FIG. 19).

The SHC beneficial mutations combinations are numbered according towild-type AacSHC (SEQ ID No. 1).

Discussion 16

The crystal structure is useful for identifying SHC/HAC derivatives withdesirable structure/activity relationships especially in relation to theconversion of homofarnesol to (−)-Ambrox. A useful pre-selection stepmight be to restrict the selection to amino acid residues located indomain 2 of the SHC/HAC crystal structure (see FIGS. 19 and 20).

Example 17

Preparation of Homofarnesol

Methods 17

General Analytical Conditions

Non-polar GC/MS: 50° C./2 min, 20° C./min 200° C., 35° C./min 270° C.GC/MS Agilent 5975C MSD with HP 7890A Series GC system. Non-polarcolumn: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm×0.25mm×12 m. Carrier Gas: Helium. Injector temperature: 230° C. Split 1:50.Flow: 1.0 ml/min. Transfer line: 250° ° C. MS-quadrupol: 106° C.MS-source: 230° C.

A) Preparation of MNU in THF

A solution of urea (175 g, 2.9 mol) and methylamine hydrochloride (198g, 2.9 mol) in water (400 ml) is heated at reflux (105° C.) for 3.5 hunder stirring. At 40° C. NaNO₂ (101 g, 1.45 mol) dissolved in water(200 ml) is added. After 15 min THF (1000 ml) is added which results ina transparent 2-phase mixture. Conc. H2SO4 (110 g, 1.1 mol) is added at0-5° C. and stirring within 1.5 h. After another 0.5 h at 0-5° C. thetwo transparent phases are separated at 25° C. The organic phase (A)(1065 ml, theoretically 1.35M) is stored for a few days at 0-5° C. orforwarded immediately to the cyclopropanation reactor.

After phase separation the water phase is extracted twice with THF(2×1:1). This gives 1100 ml of phase B and 1075 of phase C. Whereasphase A gives a 51% conversion of a terminal alkene to a cyclopropane ina subsequent cyclopropanation reaction, phase B gives <0.5% cyclopropaneand phase C gives no detectable conversion. We conclude that >99% MNU isextracted after the first phase separation. Usually the water phase istherefore discarded after the first phase separation (from organic phaseA) after treatment with cone, aqueous KOH and acetic acid.

B) Preparation of E-Δ-Farnesene Using MNU in THF

N-Methyl-N-nitroso urea 1.35M in THF (136 ml, 184 mmol) is addeddropwise at 0° C. to a rapidly stirred mixture of E-beta-Farnesene (CAS18794-84-8) (25 g, 122 mmol) and aqueous KOH (50 ml, 40%) at 0-5° C.After the addition of 4 ml of the MNU solution, Pd(acac)2 (7.4 mg, 0.024mmol, 0.02%) pre-dissolved in 0.5 ml dichloromethane is added.

The remaining MNU solution is added over 4 h at 0-5° C. A GC at thisstage showed 28% unconverted E-β-Farnesene, 65% of the desiredmonocyclopropropane (shown above) and 3% of a biscyclopropanatedcompound 5. After 16 h at 25° C. acetic acid (100 ml) is added at 0-5°C., then tert-butyl methyl ether (250 ml). After phase separation theorganic phase is washed with 2M HCl (250 ml) and the aqueous phaseextracted with tert-butyl methyl ether (250 ml). The combined organiclayers are washed with water (2×100 ml), aqueous 10% NaOH (2×100 ml) andwater (2×100 ml), dried over MgSO₄, filtered and concentrated to give26.9 g of a slightly yellow liquid which contains 9% E-β-Farnesene, 82%of the desired monocyclopropane compound and 6% of a biscyclopropanatedside product.

The desired compound could be further isolated by distillativepurification. Addition of 1 g K₂CO₃ (1 g) and distillation over a 30 cmsteel coil column at 40-60 mbar gives 147 g monocyclopropane compound(68% corr) at 135-145° C. The fractions are pooled to give 92 gmonocyclopropane compound of 100% purity.

Analytical Data of E-Δ Farnesene:

1H-NMR (CDCl₃, 400 MHz): 5.1 (2 m, 2H), 4.6 (2H), 2.2 (2H), 2.1 (4H),2.0 (2H), 1.7 (s, 3H), 1.6 (2 s, 6H), 1.3 (1H), 0.6 (2H), 0.45 (2H) ppm.13C-NMR (CDCl₃, 400 MHz): 150.9 (s), 135.1 (s), 131.2 (s), 124.4 (d),124.1 (d), 106.0 (t), 39.7 (t), 35.9 (t), 26.7 (t), 25.7 (q), 17.7 (q),16.0 (d), 6.0 (t) ppm. GC/MS: 218 (2%, M+), 203 (5%, [M−15]+), 175(11%), 147 (31%), 134 (15%), 133 (20%), 121 (12%), 107 (55%), 95 (16%),93 (30%), 91 (20%), 82 (11%), 81 (33%), 79 (42%), 69 (100%), 67 (22%),55 (20%), 53 (21%), 41 (75%). IR (film): 3081 (w), 2967 (m), 2915 (m),2854 (in), 1642 (m), 1439 (m), 1377 (m), 1107 (w), 1047 (w), 1018 (m),875 (s), 819 (m), 629 (w). Anal. calcd. for C16H26: C, 88.00; H, 12.00.Found: C. 8780; H, 12.01.

C) Preparation of (7E)-4,8,12-trimethyltrideca-3,7,1-trien-4-ol((7E)-homofarnesol)

A mixture of (E)-(6,10-dimethylundeca-1,5,9-trien-2-yl)cyclopropane (E-ΔFarnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internalstandard) and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure tube isheated under stirring at 150° C. After 18 h and complete conversion(according to GC) the mixture is poured on water (50 ml) and toluene (50ml).

The phases are separated and the aqueous phase extracted with toluene(50 ml). The combined organic layers are washed with cone, aqueousNa2CO3 (50 ml) and cone. NaCl (2×50 ml), dried over MgSO4, filtered andevaporated under reduced pressure to give a brownish resin (1.35 g)which is mixed with 30% aqueous KOH (4.3 ml) and stirred at 25° C. for 2h. GC analysis reveals formation of 96%(7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to the internalstandard. EZ ratio 68:22. The analytical data of the E-isomer areconsistent with the ones from the literature, see for example P.Kocienski, S. Wadmnan J. Org. Chem. 54, 1215 (1989).

Results 17

The data demonstrates the preparation of homofarnesol which is suitablefor bioconversion to (−)-Ambrox.

Discussion 17

This process for the preparation of homofarnesol is also described indetail in two co-pending patent applications—PCT/EP2014/072882(WO2015/059290) and PCT/EP2014/072891 (WO2015/059293)—the entirecontents of which are incorporated herein by reference.

Example 18

One Pot Reaction

In this experiment: (i) a fermentation with an E. coli strain producingthe 215G2 SHC variant (eg. as described in Example 1) followed by (ii)EEH conversion directly in the fermentation broth was carried out.Because the 3 parameters [cells], [EEH] and [SDS] (g/l) are linked, itwas required to adjust the parameters [EEH] and [SDS] in the availablevolume of fermentation broth depending on the concentration of the cells(g/l) obtained at the end of the fermentation. The target was to convert125 g/l EEH with 250 g/l cells at an SDS concentration of 1.55%. Toallow for a proper bioconversion, the cells must be in a resting state,in a status of glucose depletion. Aeration was switched off.

Method 18

Fermentation:

In order to allow for a quite accurate determination of the volume ofthe fermentation broth in the reactor at the end of the fermentation,the volumes of withdrawn samples as well as the volumes of all additionsmade to the fermenter (feed, base, acid, . . . ) were recorded.

Determination of Cell Concentration in the Fermentation Broth:

A sample of fermentation broth (5-10 ml) was drawn under constantagitation for cell wet weight (g/l) determination and placed into acentrifuge tube. Sample mass was recorded. The sample is centrifuged for10 min at 17210 g and 4° C. (eg. 12000 rpm, SS-34 rotor, Sorvall RC3Bcentrifuge). The supernant was withdrawn with careful pipetting and themass of the pellet was recorded. The cell wet weight concentration isdetermined was g_(cells)/l_(broth) or g_(cells)/g_(broth).

The volume of the fermentation broth in the fermenter was determinedaccording to all additions and withdrawals. In case the fermenter was ona scale, the mass of the fermentation broth was determined by weighing,if not it was assumed that 1 ml=1 g.

Determination of Required Homofarnesol and SDS Quantities:

According to the determined cell concentration and volume of thefermentation broth the amount of EE-Homofarnesol and SDS to add to thereactor was determined in order to keep the same ratio between the 3 asset out in the bioconversion described in Example 9: 125 g/l EEH, 250g/l cells, 1.55% SDS.

Setting Up the Bioconversion:

-   1. Temperature was set to 35° C. Aeration is switched off.-   2. To the fermentation broth was added the calculated amount of    Homofarnesol.-   3. The required amount of SDS was carefully added from an aqueous    15.5% SDS stock solution.-   4. The reaction was mixed well for approx. 15 min at 800 rpm.-   5. The pH of the reaction was recorded (internal pH electrode).-   6. A sample (approx. 1 ml) was drawn to a 15 ml Falcon tube. Approx.    5 ml deionized water was added and the pH was recorded at an    externally calibrated electrode after thorough mixing.-   7. The pH in the reactor was set stepwise to 5.4 (value measured at    the externally calibrated electrode) using 85% H₃PO₄ while regularly    controlling pH at the external electrode was described above (6.).-   8. pH was regulated during bioconversion using eg. 10-25% H₃PO₄ and    32% NaOH.-   9. Reaction sampling: approx. 1 ml reaction mixture was placed in a    15 ml falcon tube. Approx. 5 ml MTBE is added. The sample was    extracted with vigorous-   10. shaking. An aliquot was centrifuged in a tabletop centrifuge for    1 min at maximum speed (Eppendorf tube). 100 μl of the solvent phase    was added to a GC vial containing 900 μl MTBE. Samples were taken    every 1-1.5 hours during the first day of bioconversion. The    following days only 3 samples a day were taken.-   11. 1 μl of the solvent phase was analyzed for its Ambrox and EEH    content as described in Example 8.-   12. EEH conversion (%) is calculated as    100×(Ambrox_(area)(Ambrox_(area)+EEH_(area))).

Result 18

The results demonstrate that a one-pot fermentation+EEH conversion wascarried out in a KLF2000 reactor (Bioengineering) at a scale of 1.9litres. 251 g/l cells allowed conversion of 238 g EEH (25 g/l cells) to≥93% in 47 hours. When measured 93 h after start, conversion was 99%.

A similar one-pot experiment was run in an Infors HT 0.75 l reactor.After a fermentation that followed a standard protocol (Example 1) thereactor cells that had been collected from other fermentations run inparallel with the same protocol were added. The resulting broth volumewas 479 g. The cell concentration was determined as 313.7 g/l, which was1.25× the concentration of cells in a regular bioconversion (250 g/lcells). EEH and SDS were added accordingly to the reactor. 75.1 g EEH(equivalent to 157 g/EEH in this Example) were converted to 98% in lessthan 90 h. This result demonstrates that it is possible to run a one-potfermentation+EEH conversion at ≥125 g/l EEH as long as the fermentationrun provides cells that have produced the 215G2 SHC variant at a highenough cell density.

Discussion 18

Advantageously, 99% conversion of the substrate was obtained which isvery commercially useful when expensive starting material (eg. EEH) areused.

Example 19

Increasing Volumetric Productivity.

Method 19

In order to increase further the volumetric productivity a 1.5×concentrated bioconversion containing 375 g/l of cells, 188 g/l EEH,2.33% SDS was run. A regular bioconversion was run in parallel at 125g/l EEH, 250 g/l cells, 1.55% SDS (Example 7). The 2 reactions were runin Infors HT 0.750 l reactors, all other parameters being unchanged.

Result 19

The results in FIG. 22 demonstrate that the percent conversion 75 hafter start was 88 in the 1.5× bioconversion vs. 95% in the regularbioconversion. The percent conversion 96 h after start was 93% p EEHconvert in the 1.5× bioconversion vs. 97% in the regular bioconversion.The percent conversion in the 1.5× bioconversion was 96% of thatobtained in the regular bioconversion. It was noted that stirring in the1.5× bioconversion became more difficult over time as the oilyHomofarnesol disappeared, being replaced by solid reaction products.This may explain the slightly lower conversion level in the 1.5×bioconversion. Using a reactor equipped with a better mixing devicemight improve the EEH conversion in a 1.5× bioconversion. The resultindicate that it is possible to run bioconversions at 188 g/l EEH orhigher provided efficient mixing is achieved; stirring efficiencyappears to be the only limitation of the system.

(−)-Ambrox Productivity

The “(−)-Ambrox productivity” refers to the amount of recoverable(−)-Ambrox in grams per liter of biotransformation and per hour ofbioconversion time (i.e. time after the substrate was added). In thisregard and with reference to FIG. 22, the (−)-Ambrox productivity iscalculated as follows:

125 g/l EEH Bioconversion (250 g/l Cells)

productivity at 1.25 h: 10.3 gram per litre per hour

productivity at 8.25 h: 6.3 gram per litre per hour

productivity at 21.25 h: 4.1 gram per litre per hour

187.5 g/l EEH Bioconversion (375 g/l Cells)

productivity at 1.25 h: 12.2 gram per litre per hour

productivity at 8.25 h: 8.2 gram per litre per hour

productivity at 21.25 h: 5.5 gram per litre per hour

It can be considered that the productivity calculated at around 6-8hours after start is representative of the initial velocity of thereaction, which describes best the maximal conversion rate of thesystem.

Typical bioconversions using 125 g/l EEH with 250 g/l cells show anAmbrox productivity of between 63 and 8.5 gram per litre per hour afteraround 6-8 hours (representative of the initial velocity of thereaction).

Example 20

Replacing Reaction Buffer with NaCl Solution

Method 20

A regular bioconversion (125 g/l EEH, 250 g/l cells, 1.55% SDS) was runas described in Example 7 but replacing the citric acid buffer pH 5.4 byeither 0.5% or 0.9% NaCl, all other reaction parameters being unchanged.A bioconversion in citric acid buffer was run in parallel as a control.

Result 20

The results in FIG. 23 demonstrate that the EEH conversion rate was thesame as in the reactions run in buffer and 0.9% NaCl. The conversionrate was lower when in the reaction run in only 0.5% NaCl. The resultdemonstrates the possibility of running bioconversion in the absence ofbuffer provided accurate pH regulation and a sufficient ionic strengthare guaranteed.

Example 21

Extraction of the Solid Phase of the Reaction Broth

Given that (−)-Ambrox is not being soluble in water and is not liquid attemperatures below approx. 75° C., these properties were taken aspossible advantages to extract the product from the solid phase of thebiotransformation using either water miscible (eg. ethanol) and waterunmiscible (eg. toluene) solvents.

Method 21

200 ml reaction broth was centrifuged to separate the solid from theliquid (aqueous) phase (Sorvall GS3, 5000 rpm, 10 min, 10° C.). Thisseparated approx. 80 ml solid pellet from approx. an 120 ml liquidphase. Analysis (Gas chromatography, Example 8) of the aqueous phaseafter MTBE extraction showed that it contained not more than approx.0.3% of the (−)-Ambrox initially present in the 200 ml reaction broth.Toluene and ethanol 99% were used for extracting Ambrofix from the solidphase.

Result 21

Toluene Extraction:

80 ml solid phase was extracted 6× with 45 ml toluene (approx. ½ solidphase volume, vigorous shaking for 30 s, centrifugation (Sorvall GS3,5000 rpm, 10 min, 10° C.). The solvent phase was analyzed with GC forits (−)-Ambrox content. Over 99.5% of (−)-Ambrox initially present inthe reaction broth were extracted with 6 extractions representing atotal toluene vol. of 1.35× the initial whole reaction broth volume (200ml) or 3.4× the vol. of the solid phase. The graph in FIG. 24 shows theevolution of the extraction over the toluene washes as % of the(−)-Ambrox quantity initially present in 200 ml whole reaction broth(due to the volume ratio broth/toluene, % in the first extract goes over100%).

Ethanol Extraction:

80 ml solid phase was extracted (Infors Multifors HT, 35° C., 1000 rpm,30 min) with approx. 160 ml (2 vol.) ethanol 99%, followed bycentrifugation. Ambrox did not crystallize during the extractionprocedure. The graph in FIG. 25 shows that after 4 washes (total 640 mlEtOH, i.e. 3.2× the initial whole reaction broth volume or 8× the volumeof the solid phase), about 99% of Ambrox initially present in thereaction broth was recovered. Sufficient ethanol is required in thefirst extraction step to prevent Ambrox crystallization (solubility inethanol). When only 1 or ½ vol of the solid phase was used in the firstextraction step, a sticky paste was obtained, which was difficult tohandle and (−)-Ambrox crystallized as needles on the pellet duringcentrifugation. Temperature appeared as not being the factor responsiblefor this crystallization (extraction and centrifugation tested at roomtemperature and approx. 35° C.-40° C.).

The (−)-Ambrox concentration in the EtOH phase as well as the EtOH/waterratio of the liquid phase (residual moisture of the solid phase)appeared to be responsible for crystal formation. It was however notedthat it was possible to reduce the volume of ethanol to 1 vol of thesolid phase.

Comment 21

As (−)-Ambrox is not in the liquid phase at room temperature, itseparates with the biomass and can be extracted with an organic solvent(eg. a water miscible solvent (eg. ethanol) or a non-water misciblesolvent (eg. toluene). The centrifugation step that separates the(−)-Ambrox into the solid phase of the reaction mixture is advantageousbecause it reduces the amount of solvent required to extract (−)-Ambrox.

Example 22

Sensory Analysis

Purpose: to carry out a sensory analysis of (−)-Ambrox and theby-products (compounds II, III and IV) formed in the “crude” extract andin the “crystallised” extract.

Result 22(a)

EEH transformation results in (−)-Ambrox (compound 1) and (−)-Ambroxisomer (Compound IV).

Result 22(b)

EZH biotransformation results in a macrocyclic ether (compound II) and9b-epi-Ambrox (compound III).

Result 22(c)

A crude composition of (−)-Ambrox comprises compounds I, II, III and IVwith each % compound present in an amount of 87.1, 2.8, 2.5 and 7.6%respectively.

Result 22(d)

A composition of the selectively crystallised material (lab scale) hasthe same components present in an amount of 99.1, 0.1, 0.1 and 0.7%respectively.

The Sensory Analytical Results were as Follows:

(−)-Ambrox: OTH 0.2 ng/l (OTH is odor threshold).

Compound IV from EEH: weak, IsoE, woody, GC-TH 5-10 ng.

Compound II from EZH: “odorless” (GC-TH>500 ng) (GC-TH is the detectionthreshold).

Compound III from EZH: GC-TH about 10× higher than Ambrox (circa 2 ng).

Conclusion

The total percent of each of the 3 by-products (compounds II, III andIV) in the “crude” extract is about 3%.

The total percent of each of the 3 by-products (compounds II, III andIV) in the “crystallised” extract is about 1% (lab scale).

The sensory analysis of the 3 by-products (compounds II, III and IV)indicates a weaker odor than that from (−)-Ambrox.

In fact, the 9b-epi-ambrox (compound III) odor is about 10 fold weakerthan (−)-Ambrox suggesting that it is essentially odorless.

The sensory analysis demonstrated that the removal of one of moreby-product compounds from (−)-Ambrox can improve the odor of theremaining compound (i.e. (−)-Ambrox) even if the removed compounds areactually odorless compounds per se. That is, an Ambrox odor enhancementwas observed in the absence of compounds II, III and IV.

Example 23

Ambrox Recovery by Steam Extraction

Methods 23

Resulting Purity of the Crude (Steam Extracted) and Crystallized(−)-Ambrox

The EE:EZ 86:14 biotransformation reaction mixture was steam extractedand the reaction product crystallized as follows: The steam distillatewas collected as a biphasic mixture. The organic phase was retained andthe aqueous phase discarded. The composition of the organic phase wasanalysed by GC and the results shown in the Table 25 below (see“crude”). The organic phase was then concentrated to dryness. Ethanolwas then added to the crude, dried product and the mixture warmed untilthe product was dissolved. At room temperature water is slowly added and(−)-Ambrox crystallizes under occasional stirring and cooling in an icebath.

Results 23

Table 25 below also shows the GC analytics results for products obtainedafter the steam extraction/distillation step (“crude”) and thecrystallized product ((−)-Ambrox). The references in Table 25 to “EZH”and “EEH” refer to (3Z,7E) homofarnesol and 7E,3E homofarnesolrespectively.

Table 25 indicates that the particular starting material (EEH:EZH 86:14)produces the desired end product (−)-Ambrox and a very specific mixtureof by-products (II, IV nd III) using the WT SHC or a SHC derivative. Thedata for the selective crystallization show a strong enrichment of (−)Ambrox (I), with practically no by-products (II), (IV) or (III) beingfound in the crystallized sample. Accordingly, this EE:EZ mixtureprovides an olfactively pure (−)-Ambrox product which is selectivelycrystallised in a relatively straightforward and cost-effective matter.

TABLE 25 shows the GC analytics results for the crystallized product.Peak area (GC) Ambrox (II) (IV) (III) Ambrox EZH EEH (%) Crude 215073190376 588769 6751605 13429 14184 86.9 Crystallized 10088 8951 646259032941 0 0 99.1

Discussion 23

Steam extraction/filtration are environmentally friendly methods forisolating Ambrox because it offers a convenient solvent-free isolationof Ambrox with an associated inactivation of the biocatalyst.

Summary 23

The (−)-Ambrox produced using the bioconversion reaction may beextracted using solvent from the whole reaction mixture (eg. using anon-water miscible solvent or by steam extraction/distillation or byfiltration) or from the solid phase (eg. using a water miscible solvent)using methods which are known to those skilled in the art.

The invention claimed is:
 1. A process for preparing (−)-Ambrox or amixture comprising (−)-Ambrox, wherein a mixture of isomers comprising(3E,7E)-homofarnesol (EEH) is enzymatically converted to (−)-Ambrox or amixture comprising (−)-Ambrox, wherein the enzymatic conversion iscarried out using a squalene hopene cyclase/homofarnesol Ambrox cyclase(SHC/HAC) enzyme having homofarnesol Ambrox cyclase activity andcomprising an amino acid sequence that has with at least 90% identity toSEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4 under reactionconditions suitable for the production of (−)-Ambrox, and wherein themixture of isomers comprising EEH is selected from at least one of[(3E,7E) and [(3Z,7E)], [(3E,7E) and (3E,7Z)] or [(3Z,7E), (3E,7E) and(3E,7Z)] also designated as [EE:EZ], [EE:ZE] and [EE:EZ:ZE]respectively, wherein (−)-Ambrox is produced in admixture with at leastone or more of the by-products (II) or (IV) and (III)


2. The process according to claim 1, wherein the process usesrecombinant host cells producing the SHC/HAC enzyme.
 3. The processaccording to claim 2, wherein the process uses sodium dodecyl sulfate(SDS) with the recombinant host cells producing the SHC/HAC enzyme. 4.The process according to claim 3, wherein the SDS/cell ratio is in therange of 10:1 to 20:1.
 5. The process according to claim 1, wherein theconversion of homofarnesol to (−)-Ambrox takes place at a temperature inthe range of from 30° C. to 60° C. and a pH in the range of about 4 toabout
 8. 6. The process according to claim 5, wherein the conversion ofhomofarnesol to (−)-Ambrox takes place at a temperature of about 34° C.to about 50° C. and a pH in the range of about 5 to about 6.2.
 7. Theprocess according to claim 5, wherein the conversion of homofarnesol to(−)-Ambrox takes place at a temperature of about 35° C. to about 40° C.and a pH in the range of about 5.2 to about
 6. 8. The process accordingto claim 1, wherein the homofarnesol substrate comprises EE:EZ isomers.9. The process according to claim 1, wherein the homofarnesol comprisesan EE:EZ isomer mixture in the weight ratios selected from the groupconsisting of: 100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07;92:08; 91:09; 90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16; 83:17;82:18; 81:19; 80:20; 79:21; 78:22; 77:23; 76:24; 75:25; 74:26; 73:27;72:28; 71:29 70:30; 69:31; 68:32; 67:33; 66:34; 65:35; 64:36; 63:37;62:38; 61:39; and/or 60:40.
 10. The process according to claim 1,wherein the homofarnesol comprises an EE:EZ isomer mixture in a weightratio selected from the group consisting of: EE:EZ 92:08; EE:EZ 90:10;EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and/or EE:EZ 66:34.11. The process according to claim 10, wherein the homofarnesolcomprises EE:EZ isomer mixture in a weight ratio of 80:20.
 12. Theprocess according to claim 1, wherein the (−)-Ambrox is isolated fromthe mixture using an organic solvent or a steam extraction/distillationstep or filtration.
 13. The process according to claim 1, wherein the(−)-Ambrox is isolated from the solid phase of the mixture using anorganic solvent or a steam extraction/distillation step.
 14. The processaccording to claim 12, wherein the (−)-Ambrox is selectivelycrystallized using an organic solvent.
 15. The process according toclaim 14, wherein the (−)-Ambrox is substantially free of theby-products (II), (IV) and/or (III).
 16. The process according to claim1, wherein the enzymatic conversion is carried out using a SHC/HACenzyme with at least 90% identity to SEQ ID No.
 1. 17. The processaccording to claim 1, wherein the enzymatic conversion is carried outusing a SHC/HAC enzyme with at least 90% identity to SEQ ID No.
 2. 18.The process according to claim 1, wherein the enzymatic conversion iscarried out using a SHC/HAC enzyme with at least 90% identity to SEQ IDNo.
 3. 19. The process according to claim 1, wherein the enzymaticconversion is carried out using a SHC/HAC enzyme with at least 90%identity to SEQ ID No. 4.